Characterization of ACC deaminase - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4342/14/14_chapter 4.pdf · Characterization of ACC deaminase 125 walls or nematode structures

Characterization of ACC deaminase

124

Chapter 4 Biochemical and Molecular Biology of ACC Deaminase Production 4.1 Introduction Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-

aminocyclopropane-1-carboxylic acid (ACC) deaminase (EC 4.1.99.4), which regulates

ethylene production by metabolizing ACC (an intermediate precursor of ethylene

biosynthesis in higher plants) into α- ketobutyrate and ammonia. This pyridoxal

phoshphate enzyme was first isolated in 1978 from pseudomonas sp. strain ACP and

from the yeast Hansenula satrunus (Honma and Shimonura 1978); since then, it has been

detected in fungi and in a number of other bacteria. When ACC deaminase-containing

plant growth-promoting bacteria are bound to a plant, they act as a sink for ACC ensuring

that plant ethylene levels do not become elevated to the point.

Conceptually, PGPR can have an impact on plant growth and development in two

different ways: indirectly or directly. The indirect promotion of plant growth occurs when

bacteria decrease or prevent some of the deleterious effects of a phytopathogenic

organism by one or more mechanisms. On the other hand, the direct promotion of plant

growth by PGPR generally entails providing the plant with a compound that is

synthesized by the bacterium or facilitating the uptake of nutrients from the environment

(Glick 1995; Glick et al. 1999). Rhizosphere bacteria multiply to high densities on plant

root surfaces where root exudates and root cell lysates provide ample nutrients.

Sometimes, they exceed 100 times those densities found in the bulk soil (Campbell and

Greaves 1990). Certain strains of these plant-associated bacteria stimulate plant growth in

multiple ways: (1) they may fix atmospheric nitrogen; (2) reduce toxic compounds; (3)

synthesize phytohormones and siderophores; or (4) suppress pathogenic organisms

(Bloemberg and Lugtenberg 2001).

Research on the ‘biocontrol’ activity of rhizobacteria has seen considerable progress in

recent years. Disease suppression of soilborne pathogens includes competition for

nutrients and production of antimicrobial compounds or lytic enzymes for fungal cell


125

walls or nematode structures (Persello-Cartieaux 2003). By contrast, systemic resistance

can also be induced by rhizosphere-colonizing Pseudomonas and Bacillus species where

the inducing bacteria and the challenging pathogen remained spatially separated

excluding direct interactions (Van Loon et al. 1998; Ryu et al. 2004).

Etiolated pea seedlings are very sensitive to ethylene. The most widely renowned

example of the effect of ethylene on plant growth is the classical “triple” response in

etiolated dicot seedlings in the presence of ethylene. This effect consists of three distinct

morphological changes in the shape of seedlings, inhibition of stem elongation, increase

in stem diameter and horizontal growth (Akhtar et al. 2005; Khalid et al. 2006). This

“triple” response reaction of etiolated seedlings has been a reliable bioassay for ethylene

action (Guzman and Ecker 1990). In a previous study (Shaharoona et al. 2007), the effect

of inoculation with ACC utilizing and ethylene-producing rhizobacteria had been

compared through highly ethylene specific classical “triple” response bioassay. In this

study, the effect of inoculation with rhizobacteria having different ACC-deaminase

activities on extenuating the classical “triple” response in etiolated pea seedlings was

investigated.

ACC deaminase-containing plant growth-promoting bacteria up-regulate genes involved

with plant growth and protein production while down-regulating plant genes involved

with ethylene stress and defence signaling pathways (Hontzeas et al. 2004). The ACC

deaminase-containing plant growth-promoting bacteria, in part, alleviate the need for the

plant to actively defend itself against various environmental stresses (Hontzeas et al.

2004; Van Loon and Glick 2004). The crystal structure has been determined for the yeast

(Minami et al. 1998), and recently for the bacterial (Karthikeyan et al. 2004) ACC

deaminase enzymes; the biochemical and thermodynamic properties of the ACC

deaminase from Pseudomonas putida UW4 have been measured (Hontzeas et al. 2004).


126

ACC deaminase from bacteria, Pseudomonas sp. ACP, Pseudomonas putida and

Pseudomonas fluorescens (Glick et al. 1995), Enterobacter cloacae CAL2 and UW4

(Shah et al. 1998), Kluyvera ascorbata SUD165 (Burd et al. 1998), and yeast, Hansenula

saturnus (Honma and Shimomura 1978), and fungus, Penicillium citrinum (Jia et al.

2006) have been reported. Colonization of the plant root system by PGPRs was shown to

reduce pathogen attack directly through production of antimicrobial substances (e.g.

siderophores, β-1, 3 glucanase, chitinases, antibiotics, and cyanidric acid), and through

competition for space, nutrients and ecological niches. PGPRs also suppress pathogens

indirectly through induction of systemic resistance (Buchenauer 1998; Cattelan et al.

1999; Viswanathan and Samiyappan 2002).

This enzyme facilitates plant growth as a consequence of the fact that it sequesters and

cleaves plant produced ACC, thereby lowering the level of ethylene in the plant. In turn,

decreased ethylene levels allow the plant to be more resistant to a wide variety of

environmental stresses, all of which induce the plant to increase its endogenous level of

ethylene; stress ethylene exacerbates the effects of various environmental stresses. The

ACC deaminase-containing soil bacteria decrease a significant portion of the

physiological damage to plants following environmental stresses including

phytopathogen infection, exposure to extremes of temperature, high salt, flooding,

drought, exposure to metals and organic contaminants, and insect predation. For many

plants a burst of ethylene is required to break seed dormancy but, following germination,

a sustained high level of ethylene can be inhibitory to root elongation. plant growth

promoting bacteria that contain the enzyme ACC deaminase, when bound to a plant root

or to the seed coat of a developing seedling, may act as a mechanism for insuring that the

ethylene level within the plant’s tissues does not become elevated to the point where root

(or shoot) growth is impaired. By facilitating the formation of longer roots and shoots,

these bacteria may enhance the survival of some seedlings, especially during the first few

days after the seeds are planted.


127

4.1.1 Ethylene Biosynthesis in higher plants

Ethylene which is produced in almost all plants, mediates a range of plant responses and

developmental step. Ethylene is involved in seed germination, tissue differentiation,

formation of root and shoots primordial, root elongation, lateral bud formation, flowering

initiation, anthocyanin synthesis, flower opening and senescence, fruit ripening and

degreening, production of aroma, leaf and fruit abscission and response of plant to biotic

and abiotic stresses. (Saraf and Tank 2005). Ethylene is a potent plant growth regulator

that affects diverse developmental processes, including fruit ripening, senescence, and

stress responses (McKeon and Yang 1987; Reid 1987). Its role in promoting climacteric

fruit ripening is particularly well established. Chemical inhibitors of ethylene synthesis or

action completely block ripening in fruits and senescence in flowers of many plant

species.

At a molecular level, ethylene is known to induce expression of a number of genes

involved in ripening (Lincoln and Fischer 1988) and pathogen response (Ecker and Davis

1987). In some instances, ethylene is stimulatory while in others it is inhibitory. When

plants are exposed to conditions that threaten their ability to survive, the same mechanism

that produces ethylene for normal development instead produces “stress ethylene” which

may be defined as an acceleration of ethylene biosynthesis associated with biological and

environmental stresses, and pathogen attack (Abeles et al. 1992; Hyodo 1991; VanLoon

1984). Ethylene is synthesized from S-adenosylmethionine by way of the intermediate 1-

aminocyclopropane-1- carboxylic acid (ACC) (McKeon and Yang 1987).

While working on the ethylene biosynthesis pathway, Adams and Yang (1979) found that

when ACC was applied to various plant organs, an increase in ethylene production was

obtained. From their observations, ACC, as a key intermediate that linked the methionine

cycle and ethylene biosynthesis, was deemed to be the direct precursor of ethylene

production with its level directly controlling ethylene synthesis in plants.


128

Ethylene biosynthesis consists of three steps 1) L-methionine is converted to S-adenosyl

L-methionine (AdoMet), a reaction catalyzed by methionine S-adenosyl transferase.

AdoMet is also utilized in other cellular reactions such as ethylation and polyamine

synthesis. 2) The conversion of AdoMet to ACC which is catalyzed by ACC synthase

(fig. 4.1). The ACC synthase step is considered to be the rate-limiting step in the

pathway. 3) ACC is further metabolized to ethylene, carbon dioxide and cyanide by ACC

oxidase.

Since all plants respond differently to stress, it has been difficult to detail the functioning

of stress ethylene. Increased ethylene levels in plants exposed to various types of stress

like heat, wounding, pathogen infection, salt, metals and nutritional stress, with increased

damage as the result has been documented.

4.1.2 Characteristics of ACC Deaminase enzyme

Enzymatic activity of ACC Deaminase is assayed by monitoring the production of either

ammonia or α-ketobutyrate, the products of ACC hydrolysis. ACC deaminase has been

found only in microorganisms, and there are no microorganisms that synthesize ethylene

via ACC (Fukuda et al. 1993). ACC Deaminase is a multimeric enzyme (homodimeric or

homotrimeric) with a subunit molecular mass of approximately 35-42 kDa. It is a

sulfhydral enzyme in which one molecule of the essential co-factor pyridoxal phosphate

(PLP) is tightly bound to each subunit. Interestingly, this enzyme is cytoplasmically

localized so that the substrate ACC must be exuded by plant tissues and subsequently

taken up by an ACC deaminase-containing microorganism before it is cleaved (Glick

1998).

Km values of ACC deaminase for ACC has been estimated at pH 8.5, in all instances

examined, to be approximately 1.5-17.4 mM indicating that the enzyme does not have a

particularly high affinity for ACC (Honma and Shimomura 1978).


Moreover ACC levels in plants are typically in μM range, therefore in most plant tissues

the ACC concentration will be dramatically below the Km of ACC deaminase for this

substrate so that based on the Michaelis- Menton rate equation for enzyme catalyzed

reaction a small increase in the ACC concentration will result in a parallel increase in the

rate of ACC cleavage.

Figure 4.1: Pathway of ethylene biosynthesis from the methionine cycle in higher plants.

Modified figure adapted from the source ref Li (1999).

129


130

4.2 Crystal Structure of 1-Aminocyclopropane-1-Carboxylate Deaminase

PLP-dependent enzymes catalyze many important reactions that act upon amino acids,

including transamination, decarboxylation, β,γ-replacement/elimination, and

racemization. In all of these reactions (except in the case of the glycogen phosphorylase

family), the two basic chemical properties of the PLP are conserved; it forms an external

aldimine between its aldehyde group and the a-amino group of the substrates and

withdraws electrons from the substrate by serving as an electron sink.

As a PLP-dependent enzyme, the ACCD’s ring opening reaction starts with a

transformation reaction from an internal aldimine between the PLP and the enzyme to an

external aldimine. These enzymes have been classified based on their three dimensional

structure, into four folding types: 1) tryptophan synthase, 2) aspartate aminotransferase,

3) D-amino acid aminotransferase and 4) alanine racemase. In most of the PLP-

dependent enzymes, the next step is the nucleophilic abstraction of the α-substituent,

either an α-proton or a carboxylate group, to form an α-carbanionic intermediate. This

reaction mechanism cannot be applied to ACCD because the substrate (ACC) does not

contain α-hydrogen and the carboxyl group is retained in the product.

Therefore, the ring-opening reaction of ACC must be initiated without obvious

accessibility to an α-carbanionic intermediate, which is, for PLP-dependent enzymes, the

common entry for catalysis. One proposed reaction mechanism is the nucleophilic

addition to Cγ followed by the cleavage of the Cα-Cγ bond and β-proton abstraction.

Because PLP acts as an electron sink, external aldimine is fairly electrophilic, and the

nucleophilic addition to Cγ to rupture the cyclopropane ring of ACC is mechanistically

feasible.


Strain

ACC deaminase activity (nM αKB mg-1h-1)

Reference(s)

Achromobacter xylosoxidans A551 400 ± 4 Belimov et al. 2001, 2005 Achromobacter xylosoxidans Bm1 90 ± 4 Belimov et al. 2001, 2005 Achromobacter sp. strain CM1 130 ± 3 Belimov et al. 2001, 2005 Acidovorax facilis 4p-6 3,080 ± 120 Belimov et al. 2001, 2005 Azospirillium brasilense Cd1843 0 Holguin et al. 2003 Enterobacter aerogenes CAL3 16 ± 12 Shah et al. 1998 Pseudomonas putida UW4 3,030 ± 60 Hontzeas et al. 2004 Pseudomonas syringae GR12-2 3,470 ± 30 Belimov et al. 2001, 2005 Pseudomonas brassicacearum Am3 5,660 ± 12 Belimov et al. 2001, 2005 Pseudomonas putida BM3 3,780 ± 32 Belimov et al. 2001, 2005 Pseudomonas marginalis DP3 4,054 ± 27 Belimov et al. 2001, 2005 Rhizobium leguminosarum128C53K 5 ± 1 Belimov et al. 2001, 2005 Rhizobium hedysari ATCC 43676 20 ± 0.1 Ma et al. 2003 Rhizobium leguminosarum 99A1 8 ± 3 Ma et al. 2003 Rhodococcus sp. strain Fp2 7,320 ± 400 Belimov et al. 2001, 2005 Rhodococcus sp. strain 4N-4 12,970 ± 440 Belimov et al. 2001, 2005 Serratia quinivirans SUD165 12 ± 15 Belimov et al. 2001, 2005 Variovorax paradoxus 3P-3 3,700 ± 90 Belimov et al. 2001, 2005 Variovorax paradoxus 5C-2 4,322 ± 100 Belimov et al. 2001, 2005 Variovorax paradoxus 2C-1 3,588 ± 26 Belimov et al. 2001, 2005 Pseudomonas putida ATCC 17399 - Shah et al. 1998 Schizosaccharomyces pombe - Wood et al, 2002 Hansenula saturnus - Honma and shimomura,

1978

Minami R et al, 1998 Penicillium citrinum - Jia et al, 2006 Yersinia pestis - Parkhill et al, 2001 Caulobacter crescentus - Nierman et al, 2001 Bacillus anthracis - Read et al, 2002 Mesorhizobium loti - Sullivan et al, 2002 Burkholderia fungorum - NCBI microbial genome

annotation project

Table 4.1 List of ACC deaminase producing microorganisms

131


132

4.3 Mechanism of ACC Deaminase Action

A model is proposed to explain how ACC deaminase-containing plant growth promoting

bacteria can lower plant ethylene levels and in turn stimulate plant growth (Glick et al.

1998), especially under stress conditions. In this model, the plant growth-promoting

bacteria bind to the surface of either the seed or root of a developing plant; in response to

tryptophan and other small molecules in the seed or root exudates, the plant growth-

promoting bacteria synthesize and secrete the auxin, Indoleacetic acid (IAA), some of

which is taken up by the plant. This IAA together with endogenous plant IAA can

stimulate plant cell proliferation and elongation, or it can induce the activity of ACC

synthase to produce ACC (Penrose and Glick 2001) (fig. 4.2).

Some of the plant’s ACC will be exuded along with other small molecules such as sugars,

organic acids and amino acids. The exudates may be taken up by the bacteria and utilized

as a food source of the rhizosphere bacteria. ACC may be exuded together with the other

components of the root or seed exudates. ACC may be cleaved by ACC deaminase to

form ammonia and α-ketobutyrate, compounds that are readily further metabolized by the

bacteria. The presence of the bacteria induces the plant to synthesize more ACC than it

would otherwise need and also, stimulates the exudation of ACC from the plant (some of

which may occur as a consequence of plant cell wall loosening caused by bacterial IAA).

Thus, plant growth promoting bacteria are supplied with a unique source of nitrogen in

the form of ACC that enables them to proliferate/survive under conditions in which other

soil bacteria may not readily flourish. And, as a result of acting as a sink for ACC and

lowering its level within the plant, the amount of ethylene that is produced by the plant is

also reduced. Thus, the inhibition of plant growth by ethylene (especially during periods

of stress) is decreased and these plants generally have longer roots and shoots and greater

biomass (fig. 4.3).


Figure 4.2 The enzymatic reaction catalyzed by ACCD. Modified figure adapted from the

source ref Ose et al. (2003)

133


Figure 4.3 The ACC deaminase in PGPR degrades the ethylene precursor ACC. The

ACC deaminase in PGPR lowers ethylene level in plants by degrading ACC to ammonia

and α- Ketobutyrate. Lowering ethylene in plants can alleviate stress and thereby improve

plant growth. Some PGPR can also produce plant regulator IAA and further stimulate

plant growth Modified figure adapted from the source Glick and Pasternak (2003).

4.4 Materials and method: 4.4.1 Isolation of bacterial strains that contain ACC deaminase

One gram of soil was added to 50 ml sterile medium containing (per litre) 10 g proteose

peptone, 10 g casein hydrolysate, 1.5 g anhydrous MgSO4, 1.5 g K2HPO4 and 10 ml

glycerol (PAF medium) in a 250-ml flask. The flask and its contents were incubated in a

shaker (200 r.p.m.) at a temperature 300C. After 24 h, a 1-ml aliquot was removed from

the growing culture, transferred to 50 ml of sterile PAF medium in a 250-ml flask and

incubated at 200 r.p.m. in a shaker for 24 h, at 300C, the same temperature as the first

incubation.

134


135

Following these two incubations, the population of Pseudomonads and similar bacteria

(such as Enterobacter spp.) is enriched and the number of fungi in the culture is reduced.

A 1 ml aliquot was removed from the second culture and transferred to a 250 ml flask

containing 50 ml sterile minimal medium, DF salts (Dworkin and Foster 1958) salts per

litre: 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4.7H2O, 2.0 g glucose, 2.0 g gluconic

acid and 2.0 g citric acid with trace elements: 1 mg FeSO4.7H2O, 10 mg H3BO3, 11.19

mg MnSO4.H2O, 124.6 mg ZnSO4.7H2O, 78.22 mg CuSO4.5H2O, 10 mg MoO3, pH 7.2

and 2.0 g (NH4)2SO4 as a nitrogen source. Following an incubation of 24 h 1-ml aliquot

was removed from this culture and transferred to 50 ml sterile DF salts minimal medium

in a 250-ml flask containing 3.0 mM ACC (instead of (NH4)2SO4) as the source of

nitrogen. A 0.5M solution of ACC (Sigma-Aldrich Co., Mumbai, India), which is labile

in solution, was filter-sterilized through a 0.2-mm membrane and the filtrate collected,

aliquoted and frozen at – 30 0C. Just prior to inoculation, the ACC solution was thawed

and a 300- μl aliquot was added to 50ml sterile DF salts minimal medium; following

inoculation, the culture was placed in a shaker for 24 h at 300C. Dilutions of this final

culture were plated onto solid DF salts minimal medium and incubated for 48 h at 300C.

Only single isolated colonies were selected for further testing. Each selected colony is

tested for plant growth stimulation properties and ACC deaminase activity.

4.4.1.1 Isolation of acdS (ACC deaminase) Gene by PCR amplification

Genomic DNA of the bacterial isolates was isolated using Zymo research genomic DNA

Isolation kit (USA). Screening of isolates for the presence of acdS gene was done using

primer pair acdS 5′-GGCAAGGTCGACATCTATGC-3′ and 5′-

GGCTTGCCATTCAGCTAT-3′ (Duan et al. 2009). The reaction conditions for PCR

involved initial denaturation for 3 min at 950C; 30 cycles, each consisting of denaturation

for 1 min at 950C, primer annealing for 3 min at 50.80C, and extension at 720C for 4 min

and a final elongation step of 5 min at 720C.


136

PCR reactions were carried out in 25 µl reaction mixture containing 10x buffer (with 2.5

mmol l-1 MgCl2), 2.5 µl; 2 mmol l-1 dNTP mixture, 3.0 µl; 10 pmole forward primer, 0.2

µl; 10 pmole reverse primer, 0.2 µl; 2.5 mmole l-1 MgCl2, 0.2 µl; Taq DNA polymerase (1

U), 0.5 µl; Nuclease free water H2O, 16.4 µl; and 25 ng of template DNA, 1.5 µl. PCR

product was analyzed for its expected size and purity by electrophoresis on 1% agarose

gels stained with ethidium bromide.

4.4.2 Enzyme extracts preparation

ACC deaminase enzyme activity (EC 4.1.99.4) was assayed (Penrose and Glick 2003)

according to a modification of the method of (Honma and Shimomura 1978), which

measures the amount of α-ketobutyrate produced when the enzyme ACC deaminase

cleaves ACC. The bacteria were cultured first in rich medium (Tryptic Soybean Broth, Hi

media Laboratories, Mumbai, India) and then transferred to minimal medium DF

(Dworkin and Foster 1958) with ACC as the sole source of nitrogen. Bacterial cells were

grown to mid-up to late-log phase. Then cells were harvested by centrifugation, washed

with 0.1 mol L-1 Tris-HCl (pH 7.6), and incubated overnight in minimal medium

containing ACC (final concentration 3.0 mmol L-1) as sole source of nitrogen. The

bacterial cells were collected, resuspended in 0.1 M Tris-HCl (pH 8.5), and washed as

above three times. Thirty microliters of toluene was added to the cell suspension and

homogenized at the highest setting for 30s and a 100-μl aliquot of the toluenized cells

was set aside and stored at 40C for protein assay by Bradford’s method (Cat No. 105570,

Bangalore genei, India) at a later time. The remaining toluenized cell suspension was

used for assay of ACC deaminase activity.

4.4.3 Purification steps

4.4.3.1 Ammonium sulphate precipitation:

For ammonium sulphate precipitation 8 gram of ammonium sulphate was dissolved in

each 20 ml of crude extract (toluenized cell suspension) with stirring.


137

The mixture was allowed to stand for a few hour (2-3 h) followed by centrifugation at

27,000 g for 20 min. at 40C. The precipitate obtained was dissolved in 5.5 ml of a 0.1 M

potassium phosphate buffer pH 7.5 and then assayed for enzyme activity.

4.4.3.2 Ion-exchange chromatography:

Attempt to purify the enzyme was done using DEAE sephadex A-50 (anion exchange

resin) in a buffer medium (0.05 M potassium phosphate, pH 7.5 containing 1mM EDTA

and 1mM merceptoethanol) that was used to pack a column (34x1cm). Three ml of the

ammonium sulphate precipitate enzyme was then loaded into the column and allowed to

equiliberate. The column was then eluted with buffer [0.05 M potassium phosphate buffer

(pH 7.5) containing 1mM EDTA and 1mM merceptoethanol] until 16 fractions were

collected and then with buffer containing NaCl at concentration that varied from 0-0.4 M

forty fractions were collected (1.3 ml each) and assayed for enzyme activity and protein

content. Those fractions which showed enzyme activity were then pooled to give 16 ml

total volume (Palmer et al. 2007).

4.4.4 1-Aminocyclopropane-1-carboxylic acid deaminase assay

4.4.4.1 Preparation of standard

The quantity of nmol of α-ketobutyrate produced was determined by comparing the

absorbance at 540 nm of a sample to a standard curve of α-ketobutyrate ranging between

0.1 and 1.0 nmol. A stock solution of 100 mmol l-1 a-ketobutyrate (Sigma-Aldrich Co.,

Mumbai, India) was prepared in 0.1 mol l-1 Tris– HCl (pH 8.5) and stored at 40C. Just

prior to use, the stock solution is diluted with the same buffer to make a 10 mmol l-1

solution from which a standard concentration curve is generated. Each in a series of

known α-ketobutyrate concentrations is prepared in a volume of 200 μl and 300 μl of the

2,4-dinitrophenyl hydrazine reagent (0.2% 2,4-dinitrophenyl hydrazine in 2 mol l-1 HCl)

(Sigma-Aldrich Co.) was added, and the contents were vortexed and incubated at 300C


138

for 30 min. The color was developed by the addition of 2.0 ml 2 mol l-1 NaOH and the

readings were taken at 540 nm (Shimadzu UV-1800, Japan).

4.4.4.2 ACC Deaminase Assay

All sample measurements were carried out in triplicate. Two hundred microlitres of the

toluenized cells were placed in a fresh 1.5 ml microcentrifuge tube and 20 μl of 0.5 mol

l-1 ACC was added to the suspension, briefly vortexed and then incubated at 300C for 15

min. Following the addition of 1 ml of 0.56 mol l-1 HCl, the mixture was vortexed and

centrifuged for 5 min at 12 000 g at room temperature. One millilitre of the supernatant

was vortexed together with 800 μl of 0.56 mol l-1 HCl. Then, 300 μl of the 2, 4

dinitrophenyl hydrazine reagent was added to the glass tube, the contents vortexed and

then incubated for 30 min at 300C. Following the addition and mixing of 2 ml of 2 N

NaOH, the absorbance was measured at 540 nm (Shimadzu UV-1800, Japan) (Penrose

and Glick 2003).

4.4.5 FTIR analysis and UV-Vis. spectra for determination of α-ketobutyric acid

synthesis

The Fourier transform infrared (FT-IR) spectra were recorded on a BRUCKER-

TENSOR-27 FTIR spectrometer as a KBr pellets. The UV-Vis. Spectra were recorded on

shimadzu UV-1800.

4.4.6 Optimization of enzyme activity at different stress conditions

4.4.6.1 Effect of ACC concentration on rate of reaction

The enzyme assay was carried out as usual except that the concentration of ACC was

varied from 0-2.5 mM under optimum pH and temperature condition. The final reaction

volume of 1.0 ml was maintained by adjusting with double distilled water. A plot of 1/V

vs. 1/[S] was used to determine the Km and Vmax of the enzyme.


139

4.4.6.2 Effect of pH

pH studies were carried out ranging from pH 1-10. To determine the optimum pH for the

enzyme activity is highest.

4.4.6.3 Effect of temperature

Enzyme assay was carried out at temperature ranged between 40C-600C. Once the

desired temperature was reached, the substrate was then added and incubation was done

for 30 min. After which test for enzyme activity was carried out as above given enzyme

assay method.

4.4.6.4 Effect of metal ions

Influence of various metal ions on ACC deaminase production was determined by

incubating the eluted aliquots from ion exchange column for 60 minutes at room

temperature. After the incubation the enzyme activity was checked in all samples.

4.4.6.5 Effect of salt concentrations

To determine the activity of ACC deaminase under high salinity different concentration

of NaCl was used ranged 10 ppm to 80 ppm. And the enzyme activity was carried out at

each concentration by above given method.

4.4.7 Characterization for Plant growth promoting potentials

4.4.7.1 Phosphate solubilization and phytase assay

The plates were prepared with Pikovaskya’s medium. The isolates were streaked on the

plates and incubated in an incubator at 28°C for 7 days. The solubilization haloes around

colonies and colony diameters were measured after 3, 5, and 7 days of incubation. Halo

size was determined by subtracting the colony diameter from the total diameter. The

isolates were also tested in the liquid pikovasky’s medium for quantitative phosphate


140

solubilization and decrease in the pH (Pikovaskya 1948). A modified method of (Fiske

and Subbarow 1925) was used to quantify the phytase activity.

4.4.7.2 Detection and characterization of siderophore

The siderophore production was determined by performing the chrome azurol S (CAS)

assay (Schwyn and Neiland 1987). All the glassware was cleaned with 6 N HCl. The

medium was deferrated by extracting with 3% 8-hydroxyquinoline in chloroform. The

medium was then autoclaved to remove any residual chloroform. Cultures of test strain

MSA1 and MS2 was raised in DF minimal medium at 30°C to a density of 108 CFU/ml.

Cells in late log phase were removed by centrifugation at 3000 rpm, and the filtrate was

tested for siderophore on CAS agar plates. Also, the quantitative estimation was

performed according to the method of Chambers et al. (1996). Specific tests were carried

out for identification of hydroxamate, and Catecholate types of siderophores following

the standard methods (Arnow 1937).

4.4.7.3 Determination of indole acetic acid (IAA) produced and detection of

gibberellic acid (GA3) production by thin layer chromatography

The production of indole-3-acetic acid (IAA) was determined by following Bric et al.

(1991). Selected bacterial strains were grown in glycerol-peptone broth with and without

tryptophan (500 mg ml-1) and incubated at 28 °C for 3 days. A 2-ml culture was taken

from each tube and centrifuged at 10,000 rpm for 15 min. One millilitre of the

supernatant fluid was taken to a clean dry tube to which 100 ml of 10 mM

orthophosphoric acid and 2 ml of reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4)

were added. After 25 min, the absorbance of the pink colour was measured

spectrophotometrically at 530 nm (Shimadzu UV-1800, Japan). The IAA concentration in

the culture was determined by using a calibration curve of pure IAA as a standard (Bano

and Musarrat 2003). The supernatant was acidified to pH 2.5 to 3.0 with 1 N HCl and

extracted twice with ethyl acetate at double the volume of the supernatant. Extracted

ethyl acetate fraction was evaporated to dryness in a rotatory evaporator at 40°C.


141

The extract was dissolved in 300 ml of acetone and kept at –20°C. Ethyl acetate fractions

(10-20 μl) were plated on TLC plates (Silica gel G f254, thickness 0.25 mm, Merck,

Germany) and developed either in isopropanol: ammonia: water (80:10:10) or benzene:n-

butanol:acetic acid (70:25:5). Spots with Rf values identical to authentic GA3 were

identified under UV light (254 nm) by spraying the plates with Ethanoic H2SO4 (90:10

V/V) (MacMilan and Suter 1963).

4.4.8 Identification of the organism by Biochemical tests and 16S rRNA sequencing

Identification of these two isolates were carried out by biochemical characterization and

then by 16S rRNA sequencing. Sequence data was aligned and analyzed by BLAST

analysis with the NCBI database for finding the closest homology.

4.4.8.1 Isolation and quantitative estimation of genomic DNA

Total Genomic DNA of the bacterial isolates was isolated using Zymo research genomic

DNA Isolation kit (USA). Spectrophotometer analysis was carried at 260 nm and 280 nm

to check the purity and the quantity of genomic DNA.

4.4.8.2 PCR amplification of bacterial 16S rRNA gene and sequencing

Screening of isolates for the presence of 16S rRNA was done using universal primer pair

10F 5’AGAGTTTGATCMTGGCTCAG3′ and 10R

5′TACGGHTACCTTGTTACGACTT3′. PCR reactions were carried out in 25 µl

reaction mixture containing 10x buffer (with 2.5 mmol l-1 MgCl2), 2.5 µl; 2 mmol l-1

dNTP mixture, 3.0 µl; 10 pmole forward primer, 0.2 µl; 10 pmole reverse primer, 0.2 µl;

2.5 mmole l-1 MgCl2, 0.2 µl; Taq DNA polymerase (2.5 U), 0.5 µl; Nuclease free water

H2O, 16.4 µl; and 50 ng of template DNA, 1.5 µl. DNA samples were amplified on DNA

thermalcycler (Eppendorf Master Cycler Gradient, Westbury, NY, USA). The PCR

conditions were as follows: initial denaturation for 3 min at 940C; 30 cycles, each

consisting of denaturation for 1 min at 940C, primer annealing for 1 min at 550C, and

extension at 720C for 2 min and a final elongation step of 7 min at 720C. PCR product


142

was analyzed for its expected size and purity by electrophoresis on 1% agarose gels

stained with ethidium bromide. Finally sequencing of PCR product was done by

microbial type culture collection and gene bank (MTCC), Chandigarh, India.

4.4.8.3 Culture deposited

Then these two cultures (MSA1 and MSA2) were deposited in the microbial type culture

collection and gene bank (MTCC), Chandigarh, India. Accession numbers provides by

the MTCC for MSA1 is Enterobacter cloacae MTCC 10018 and for MSA2 is

Enterobacter cancerogenus MTCC 10019.

4.4.9 Nucleotide sequence accession numbers

Sequence data reported in present study has been deposited in the GenBank nucleotide

sequence database under the accession numbers HM131220 for MSA1 and HM131221

for MSA2.

4.4.10 Seed bacterization

Jatropha curcas seeds were surface sterilized with 1% sodium hypochlorite for 5 min and

washed five times with sterilized distilled water. Seeds were coated with 1%

carboxymethylcellulose as adhesive. Liquid medium was prepared by using minimal salts

medium containing ACC as sole nitrogen source. Each strain was inoculated in 150 ml

flask containing 60 ml medium and incubated at 28 ± 10Cfor three days. An optical

density of 0.5 recorded at λ 535 nm was achieved by dilution to maintain uniform cell

density (108-109 CFU/ml).

4.4.11 Effect of bacteria on plant growth in greenhouse conditions

Five inoculated seeds of Jatropha were sown in each polybag of 15 cm diameter filled

with sandy loam soil and watered regularly. For each treatment, three such polybags were

maintained. Uninoculated seeds were sown in polybag served as control.


143

For each observation, two plants were randomly selected from each treatment and the

mean of two plants was used as one replication. The experiment was repeated twice.

Observations were recorded on rate of seedling emergence, root length, shoot length.

Chlorophyll content, leaf area, and dry mass of root, shoot, and total plant drawing

random samples at 30th, 60th , 90th , and 120 days after showing (DAS).

4.4.12 Data analysis

All glasshouse experiments were arranged in completely randomized block design with

three replications in each treatment and repeated twice. The data were subjected to

analysis of variance and mean values in each treatment were compared using least

significant differences at 5% probability (P=0.05).

4.5 Results and discussion: 4.5.1 Isolation and identification (Biochemical tests and 16S rRNA sequencing) of Bacterial cultures

Organisms those grew diffusively on the positive ACC- DF medium were identified

based on the biochemical (Table 4.2) and 16S rRNA sequencing as Enterobacter cloacae,

designated as MSA1 and Enterobacter cancerogenus, designated as MSA2. Strain

Enterobacter cancerogenus MSA2 were found Gram-negative, straight rods that are

motile with peritrichous flagella and are facultatively anaerobic. Strain MSA2 gives

positive results for following characteristics: catalase production, nitrate reduction,

Voges-Proskauer reaction, KCN tolerance, esculin hydrolysis, β-galactosidase

production, utilization of acetate, citrate, glutamate, DL-lactate, malate, succinate, L-

alanine, DL-α-alanine, and L-serine, production of acid from L-arabinose, D-xylose, D-

ribose, D-glucose, D-lactose, D-galactose, L-rhamnose, D-mannose, D-fructose, D-

trehalose, D-cellobiose, D-mannitol, glycerol, salicin, mucate, pyruvate, and α-D-

galacturonate. Liquefaction of gelatin at 27°C is evident at fifteen days.


144

The strain gave negative results for the following characteristics: pigment production,

oxidase, the methyl red test, production of deoxyribonuclease, hydrogen sulfide, lipase,

lysine decarboxylase, and urease, phenylalanine deamination, reducing substances

produced from sucrose, utilization of alginate, benzoate, propionate, and sodium

potassium tartrate; production of acid from L-sorbose, melezitose, ethanol, adonitol, i-

erythritol, inulin, glycogen, chitin, and D-tartaric acid, production of gas from D-

arabinose and myo-inositol. While strain Enterobacter cloacae MSA1 were found Gram-

negative, straight rods that are motile with peritrichous flagella and are facultatively

anaerobic. Enterobacter cloacae MSA1 was found having almost same biochemical

characteristics differences were found as MSA1 positive for reducing substances

produced from sucrose, utilization of alginate, benzoate, propionate, and sodium

potassium tartrate; production of acid from L-sorbose, melezitose, ethanol, adonitol, i-

erythritol, inulin, glycogen, chitin, and D-tartaric acid, production of gas from D-

arabinose and myo-inositol, urease test, ornithine decarboxylase and slow positive results

were obtained for gelatine utilization test while these tests were negative for MSA2. HCN

(Hydrocyanic acid) was found negative in case of MSA1 and positive in MSA2. With

these biochemical characteristics and the distinguish character like indole positive and

their plant growth promoting attributes with the Jatropha curcas plant put this culture in

the novel one and pioneer to be reported with the Jatropha plant.

PCR was carried out to specifically identify the Enterobacter strains using gene-specific

primers. The ITS primers amplified a DNA fragment of 1396 bp corresponding to the

region of 16S rRNA intervening sequence for Enterobacter sp. Two isolates were

examined for the amplification of 16S rRNA region and both the isolates amplified the

DNA product with the size of 1396 bp. The results of PCR amplification has confirmed

that these isolates were pertained to the group of Enterobacteriaceae (pic. 4.1). Finally

sequencing of PCR product was done by microbial type culture collection and gene bank

(MTCC), Chandigarh, India. Then these two cultures (MSA1 and MSA2) were deposited

in the microbial type culture collection and gene bank (MTCC), Chandigarh, India.


Accession numbers provides by the MTCC for MSA1 is Enterobacter cloacae MTCC

10018 and for MSA2 is Enterobacter cancerogenus MTCC 10019.

Picture 4.1 Amplification of Enterobacter strains using rDNA intervening sequence-specific primers. (M) 100 bp Ladder, (MSA1) Enterobacter cloacae MSA1; (MSA2) Enterobacter cancerogenus MSA2

145


146

Sequence obtained reported in present study has been also deposited in the NCBI

GenBank nucleotide sequence database under the accession numbers HM131220 for

MSA1 and HM131221 for MSA2. Phylogenetic analysis based on 16S rRNA gene

sequences available from the European Molecular Biology Laboratory data library

constructed after multiple alignments of data by ClustalX. Distances and clustering with

the neighbor-joining method was performed by using the software packages Mega

version 4.0. Bootstrap values based on 1000 replications are listed as percentages at the

branching points. An almost complete 16S rRNA gene sequence comprising 1396

nucleotides was determined and compared with representative sequences of members of

the family Enterobacteriaceae. The strain MSA1 formed a separate branch in neighbor-

joining (fig. 4.4) and was grouped most closely to a cluster containing E. cloacae with

99% similarity and MSA2 grouped most closely to E. dissolvens LMG 2683 with 99%

sequence similarity, and 98.3% similarity with the E. cancerogenus LMG 2693.

Recently, E. dissolvens was reassigned as a subspecies of E. cloacae as E. cloacae

subspecies.


147

Biochemical Characteristics Enterobacter

cloacae MSA1 Enterobacter cancerogenus MSA2

Utilization of acetate, citrate, glutamate, DL-lactate, malate, succinate, L-alanine, DL-α-alanine, and L-serine, production of acid from L-arabinose, D-xylose, D-ribose, D-glucose, D-lactose, D-galactose, L-rhamnose, D-mannose, D-fructose, D-trehalose, D-cellobiose, D-mannitol, glycerol, salicin, mucate, pyruvate, and α-D-galacturonate.

+

+

Reducing substances produced from sucrose, utilization of alginate, benzoate, propionate, and sodium potassium tartrate; production of acid from L-sorbose, melezitose, ethanol, adonitol, i-erythritol, inulin, glycogen, chitin, and D-tartaric acid, production of gas from D-arabinose and myo-inositol,

+

-

Pigment production, oxidase, hydrogen sulfide, lipase, lysine decarboxylase

-

-

Catalase production, KCN tolerance, esculin hydrolysis, β-galactosidase production,

+

+

Voges-Proskauer reaction, + + Phenylalanine deamination - - Urease test + - Methyl red test, - - Nitrate reduction test + + Arginine decarboxylase + + Gelatin utilization (+) + Indole production test - (+) Ornthine decarboxylase + - Motility + + HCN - + Table 4.2 Biochemical characterization of Enterobacter cloacae MSA1 and Enterobacter cancerogenus MSA2, +, Positive; -, Negative; (+), Slow positive


Enterobacter cloacae strain E274

Cronobacter sakazakii strain E413



Enterobacter sp. M2

Enterobacter cloacae strain 279-56

MSA1

Enterobacter sp. PR5

MSA2

Enterobacter dissolvens LMG 2683

Enterobacter cancerogenus LMG 2693

Uncultured Klebsiella sp.

Enterobacter cloacae strain B5

Enterobacter cloacae strain FR

Enterobacter aerogenes strain HK 20-1

Enterobacter aerogenes strain HC05061

Enterobacter ludwigii strain CaR

77100

97100

100100

46

36

27

26

13

40

28

100

Figure 4.4 Phylogenetic analysis based on 16S rRNA gene sequences available from the European Molecular Biology Laboratory data library constructed after multiple alignments of data by ClustalX. Distances and clustering with the neighbor-joining method was performed by using the software packages Mega version 4.0. Bootstrap values based on 1000 replications are listed as percentages at the branching points.

4.5.1.2 Quantitative estimation of genomic DNA

DNA and RNA both strongly absorb UV light with a maximum absorbance at 260 nm

and protein absorbs UV light maximally at 280 nm. Range of purity spectrum lies

between 320 nm to 220 nm. The A260/A280 ratio from a preparation of pure double

stranded DNA should be between 1.5 and 2.0. Higher ratio is often due to RNA

contamination and lower ratio indicates the presence of protein. Extinction co efficient

for protein is much lower than the extinction co efficient for nucleic acid. Ratio greater

than 0.50 is indicative of protein contamination.

148


149

Here the ratio falls between the range in both the cases and the concentration of DNA in

MSA1 was 86.76 ng/µl which is higher and pure while in MSA2 concentration was 35.08

ng/µl is moderate and shows the purity as the A260/A280 ratio falls in the range (Table 4.3).

Isolates Concentration (ng/µl)

A 260 A280 A260/A280 A260/A230

MSA1 86.76 1.735 1.077 1.61 0.09 MSA2 35.08 0.710 0.459 1.54 0.05

Table 4.3 Quantitative estimation of Genomic DNA

4.5.1.3 Isolation of acdS (ACC deaminase) Gene by PCR amplification

An ACC deaminase structural gene (acdS) was PCR amplified from both the

Enterobacter strains. The primers 5′-GGCAAGGTCGACATCTATGC-3′ and 5′-

GGCTTGCCATTCAGCTAT-3′ (Duan et al. 2009) were used to obtain acdS genes.

After PCR, acdS gene of desired product was extracted using a Qiagen min elute gel

extraction kit (Qiagen, USA) and about 1.7 kb in size identical to each other were

obtained for MSA1 and MSA2 both the strains (pic 4.2). After Gel elution again Gel for

acdS was carried to confirm and pure product of gene (pic. 4.3). Amplification of acdS

gene gives the proof for the presence of gene in the form of 1.7 kb gel band. By using the

same primer as reported earlier Duan et al. (2009) obtained acdS genes from 17 strains of

Rhizobia of 1 kb in size. ACC deaminase producing bacteria are known to facilitate the

growth of a variety of plants especially under stressful conditions such as flooding, heavy

metals, high salt and drought So, the acdS gene coding for enzyme ACC deaminase can

be a very useful candidate gene for the development of transgenics for abiotic stress

management in plants.


Picture 4.2 Amplification of ACC deaminase (acdS) gene. (M) 1 Kb Ladder, (MSA1) Enterobacter cloacae MSA1; (MSA2) Enterobacter cancerogenus MSA2

Picture 4.3 Gel elution and Gel electrophoresis for confirmation of acdS gene. (M) 1 Kb Ladder, (MSA1) Enterobacter cloacae MSA1; (MSA2) Enterobacter cancerogenus MSA2

150


151

4.5.2 Enzyme activity after ion-exchange chromatography

Figure 4.5 shows the Activity of enzyme with respect to the elution profile of ACC

deaminase from a column that was packed with DEAE sephadex (A-50). MSA1 shows

enzyme activity between the fractions 4-20 while MSA2 shows activity between fractions

6-20. Maximum activity shown by MSA1 and MSA2 at the 10th and 12th fractions and

was recorded as 0.12 and 0.14 (mM α-ketobutyrate h-1 ml-1) x 10-2 respectively.

4.5.3 Determination of α-ketobutyrate by FT-IR and UV-Vis. Spectra.

The presence of ACC deaminase was verified by FTIR (fig. 4.6 and fig. 4.7) and UV

spectra (fig. 4.8 and fig. 4.9), this confirmed that the isolates produced ACC deaminase.

FT-IR spectra clearly shows the peak at 1683 cm-1 which shows that ketonic group is

present (-C=O). Whereas 3452 cm-1 peak shows that the presences of amino group (-

NH2). Absorption maxima of both the culture was quiet similar and the characteristic

peak was found between the 400 to 500 nm which shows meaningful for α-ketobutyrate.


0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

2 4 6 8 10 12 14 16 18 20 22

Fraction No.

AC

C d

eam

inas

e ac

tivity

(m

M α

- ket

obut

yrat

e m

l-1 h

-1) x

10-

2

MSA1 MSA2

Figure 4.5 Enzyme activity after Ion-exchange chromatography using DEAE- Sephadex A-50

152


Figure 4.6 FT-IR spectra of the α- ketobutyrate from culture MSA1, lower line obtained by standard α- ketobutyrate and the upper line by the culture supernatant.

Figure 4.7 FT-IR spectra of the α- ketobutyrate from culture MSA2, lower line obtained by standard α- ketobutyrate and the upper line by the culture supernatant.

153


Figure 4.8 Absorption maxima for the culture supernatant MSA1

Figure 4.9 Absorption maxima for the culture supernatant MSA2.

154


155

4.5.4 Optimization of enzyme activity at different stress conditions 4.5.4.1 Optimum pH and temperature Various studies were done using the ACC deaminase that was partially purified. The

results from pH experiment shows that the enzyme acts within a narrow pH range (fig.

4.10). Maximum activity (318 and 220 nm α-ketobutyrate mg-1 h-1) for MSA1 and MSA2

respectively is occurring at pH 7.5. Figure 4.11 shows that initially the activity of the

enzyme increases as the temperature was increased. The enzyme activity dramatically fell

upon reaching 300C and activity was lost at 600C and beyond, a temperature optimum

was displayed at approximately 280C (320 and 223 nm α-ketobutyrate mg-1 h-1) for

MSA1 and MSA2 respectively. Hontzeas et al., (2004) reported an optimum pH and

temperature of 8.0 and 220C, respectively for ACC deaminase isolated from the

rhizobacterium pseudomonas putida (UW4). A difference in pH and temperature

optimum seems to be species related.


0

50

100

150

200

250

300

350

400

1 2 3 4 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

pH

AC

C d

eam

inas

e ac

tivity

nm

α- k

etob

utyr

ate

mg-

1 h-

1

MSA1 MSA2

Figure 4.10 Effect of pH on ACC deaminase activity.

-50

0

50

100

150

200

250

300

350

400

4 10 15 28 37 50 60

Temperature

AC

C d

eam

inas

e ac

tivity

nm

α- k

etob

utyr

ate

mg-

1 h-

1

MSA1 MSA2

Figure 4.11 Effect of temperature on ACC deaminase activity

156


157

4.5.4.2 Effect of metal ions and salt concentration

Different metal ions such as CaCl2, MgSO4, K2HPO4 and CuSO4 at a concentration of

5mM were used. Enzyme shows their activity in the presence of selected metal ions (fig.

4.12), highest activity shows by MSA1 in the presence of K2HPO4 280 nm α-ketobutyrate

mg-1 h-1 while MSA2 shows highest value in presence of MgSO4 (194 nm α-ketobutyrate

mg-1 h-1). Burd et al., (2000) reported on the potential of the ACC deaminase-producing

bacterium Kluyvera ascorbata SUD165 to protect canola (Brassica napus) and tomato

(Lycopersicon esculentum) seeds from the heavy metal toxicity induced by high

concentrations of nickel (Ni), lead (Pb) and zinc (Zn). MSA1 shows ACC deaminase

activity highest up to the 50 ppm concentration of NaCl (54 nm α-ketobutyrate mg-1 h-1)

and MSA2 gives enzyme activity highest at 10 ppm (54.3 nm α-ketobutyrate mg-1 h-1)

and then the activity decreases continuously (fig.4.13). Saravanakumar and Samiyappan

(2007) reported that Pseudomonas fluorescens TDK1 possessing ACC deaminase activity

enhanced the saline resistance of groundnuts and observed increase yields over the

groundnuts treated by Pseudomonas spp. that lacked ACC deaminase activity.

4.5.4.3 Effect of substrate concentration

A plot of 1/V vs. 1/[S] was used to determine the Km and Vmax of the enzyme. There

was a steady increase in activity with increase in substrate concentration. Results (fig.

4.14 and fig. 4.15) demonstrate a Km of 12.5 mM and a Vmax of 0.285 nm α-

ketobutyrate mg-1 h-1 in case of MSA1 isolate while in MSA2 Km of 2.08 mM and Vmax

of 0.04 nm α-ketobutyrate mg-1 h-1 of was found. Characteristic of all ACC deaminases is

their low affinity for ACC. When the Km values for the binding of ACC by ACC

deaminase were determined for enzyme extracts of several different microorganisms at

their optimum pH the values ranged from 1.5 to 17.4 mM, indicating that the enzyme

does not have a particularly high affinity for ACC.


0

50

100

150

200

250

300

350

CaCl2 MgSO4 K2HPO4 CuSO4

Enzyme + metal ions

AC

C d

eam

inas

e ac

tivity

nm

α- k

etob

utyr

ate

mg-

1 h-

1

MSA1 MSA2

Figure 4.12 Effect of metal ions on ACC deaminase activity.

0

10

20

30

40

50

60

70

10 20 30 40 50 60 70 80

NaCl concentration (mg/ml)

AC

C d

eam

inas

e ac

tivity

nm α

- ket

obut

yrat

e m

g-1

h-1

MSA1 MSA2

Figure 4.13 Effect of salt concentration on ACC deaminase activity.

158


-20-15-10-505

101520253035404550

-1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2

1/ (ACC), mM-1

1/[ n

m α

- ket

obut

yrat

e m

g-1

h-1]

Figure 4.14 Effect of ACC concentrations on ACC deaminase activity of MSA1 culture.

-20

-10

0

10

20

30

40

50

60

70

80

90

-1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2

1/ (ACC),mM-1

1/[ n

m α

- ket

obut

yrat

e m

g-1

h-1]

Figure 4.15 Effect of ACC concentrations on ACC deaminase activity of MSA2 culture

159


160

For biotechnological applications, improving upon ACC deaminase efficiency (defined as

Kcat/Km) could be achieved by increasing Kcat or, conversely, decreasing Km. In this

instance it is the latter that needs improvement. Rational design or random mutagenesis to

yield a more efficient ACC deaminase would be a worthy goal.

4.5.5 Characterization for PGP Potentials

4.5.5.1 Phosphate solubilization and phytase assay

Mineral phosphate solubilization through organic acid production appears to be the

mechanism used by most rhizospheric species, but in these isolates presumably exists an

additional mechanism of phytase production for solubilization of organic complex of

phosphates. Results shows that the isolates MSA1 and MSA2 showed zone of phosphate

solubilization on solid Pikovskyaya’s medium after 3rd day of incubation at 28 ± 2 oC.

Maximum TCP solubilization in liquid medium was observed in MSA2 (22 µg/ml) while

MSA1 solubilize 20 µg/ml. The pH of the medium also showed a decrease from 6.5 to a

maximum of 3.3 after 14 day (fig. 4.16). These isolates as showing the good inorganic

phosphate solubilizer are also able to mobilize soil organic phosphates due to presence of

phytase enzyme. MSA1 shows the phytase activity 0.402 units/ml and MSA2 shows

0.335 units/ml (fig. 4.17).

4.5.5.2 Detection and characterization of siderophore

Siderophore production by the isolates carried out on solid CAS blue agar showed a clear

zone of decolorization representing iron chelation by the isolate in the medium (fig.4.18).

The isolate MSA1 showed siderophore production (27 µgml-1) after 96 h of incubation.

Siderophore production reduced thereafter on further incubation up to 144 hour and the

isolate MSA2 produce 22 µgml-1 siderophore (fig. 4.18). Production of siderophores by

plant growth-promoting rhizobacteria is considered to be important in the suppression of

deleterious microorganisms and soil borne plant pathogens and in some cases appears to

trigger induced systemic resistance.


20

3.37

16

22

4.01

14

2

7

12

17

22

pH

Phos

phat

e S

olub

iliza

tion

(µg/

ml)

0

5

10

15

20

25

Zone

of P

hosp

hate

So

lubi

lizat

ion

(mm

)

MSA1 MSA2

Figure 4.16 Phosphate solubilization (quantitative), zone of phosphate solubilization (qualitative) and the pH changes by the culture MSA1 and MSA2.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

MSA1 MSA2

Isolates

Phyt

ase

(uni

ts/m

l)

Figure 4.17 Phytase production by the culture MSA1 and MSA2.

161


162

4.5.5.3 Determination of indole acetic acid (IAA) and gibberellic acid (GA3)

Indole acetic acid production was quantified (fig.4.19) for both the culture and it was

found that the highest concentration of IAA (32 µg/ml) were produced by bacterial strain

Enterobacter cloacae MSA1 and Enterobacter cancerogenus MSA2 produces 25 µg/ml

of IAA in the 96th hour of inoculation. Glick et al. (1998) proposed a model that explains

how ethylene and IAA interact as a feedback loop. The decrease in ethylene levels by

ACC deaminase not only down regulates the plant stress responses but also relieves the

ethylene repressed auxin responses factor (ARF) synthesis, leading to plant growth

promotion resulted from both stress alleviation and growth simulation. However, with the

increase in ARF synthesis, ACC synthase is also simulated to produce more ACC and

ethylene, which represses the ARF synthesis. In this way, ethylene limits its own

production. Evidence from thin layer chromatography showed the appearance of

gibberellins like compound in the cultures but its occurrence was not reproducible. Its Rf

(0.6) value was equivalent to that of GA3.


0

5

10

15

20

25

30

MSA1 MSA2

Isolates

Side

roph

ore

prod

uctio

n μg

/ml

02468101214161820

Zone

Siz

e (m

m)

Siderophore production Zone Size

Figure 4.18 Siderophore production (quantitative), zone of Siderophore production (qualitative) by the culture MSA1 and MSA2.

0

5

10

15

20

25

30

35

0 72 96 120

Duration (h)

IAA

pro

duct

ion

(µg/

ml)

MSA1 MSA2

Figure 4.19 Indole acetic acid production by the cultures MSA1 and MSA2.

163


164

4.5.6 Effect of bacteria on plant growth in greenhouse conditions

Significant qualitative as well as quantitative variations were observed between PGPR-

treated and non-treated control. Results regarding the effect of inoculation with

rhizobacteria on root, shoot and leaf of Jatropha are been summarized in table 4.4 and

table 4.4 and the significance test in table 4.6 and table 4.7. It is evident from that data

that inoculation with MSA1 and MSA2 caused significant plant growth promotion as

compared with the control (pic.4.4 and 4.5). Table 4.3 described the root length of

Jatropha curcas seedlings was significantly increased by inoculation with MSA1 that

shows 30.53 %, 36.39 %, 25.34 % and 23 % from 30, 60, 90 and 120 DAS over

uninoculated control. Similarly caused increase in the fresh root weight of Jatropha

seedlings over uninoculated control that shows 90.40 %, 138.43 %, 51.71 %, and 106.35

% while dry weight shows the increase from 73.17 %, 72.16%, 6.88 % to 111.43 % from

30 to 120 DAS respectively. The shoot length of Jatropha seedlings increased with the

inoculation 10.91%, 2.44%, 17.26% and 28.17 % from 30, 60, 90 and 120 DAS, similarly

fresh shoot weight increased 30.94%, 114.63%, 87.17% to 120.02 % and the dry shoot

weight increased 90.17%, 104.15%, 525.07% to 365.64 % from 30 to 120 DAS

respectively. Shoot width of Jatropha was calculated and found that 25.54%, 18.02%,

8.76% to 22.17% increased over uninoculated control from 30 to 120 DAS.

Isolate MSA1 was also effective in significantly increasing the number of leaves per

plant of Jatropha resulted in to 21.45%, 53.81%, 56.28% and 15.96% from 30,60, 90,

and 120 DAS compared with their respective uninoculated control. While the leaf length

and leaf width increased 18.49%, 66.04%, 39.97% to 27.15 % and 14.73%, 36.80%,

34.18% to 13.67 % from 30 to 120 DAS. Total chlorophyll content increased respectively

from 39.53%, 30.107%, 57.42% and 59.50 % from 30, 60, 90 and 120 DAS over the

uninoculated control. Biomass of Jatropha curcas was also increased over control after

inoculation with the culture MSA1 and resulted into 20.92%, 125.19%, 83.96% and

122.53 % from 30, 60, 90 and 120 DAS.


165

These observations may be of crucial importance for investigations aimed at isolation,

characterization, and further application of PGPR as biopreparations in agriculture that

presupposes a large-scale release of bacteria to the environment. Inoculation with

rhizobacteria containing ACC deaminase reduced the effects of water stress applied

owing to low soil moisture and, in most of the cases, significantly increased the fresh

weight and dry weight compared with their respective uninoculated control. The reduced

ethylene biosynthesis in tomato plants transformed with bacterial ACC deaminase from

Enterobacter cloacae UW4 decreased disease symptoms of Verticillum wilt (Robison et

al. 2001). In agreement with these observations that decreasing plant ethylene sensitivity

or biosynthesis can limit disease development. This hypothesis relies on previous

observations that rhizosphere inoculation with ACC deaminase containing bacteria

decreases root ACC levels and ethylene evolution (Burd et al. 1998; Penrose et al. 2001).


30 DAS 60 DAS 90 DAS 120 DAS Vegetative parameters C MSA1 C MSA1 C MSA1 C MSA1

Length (cm) 14.54 ± 0.3 18.98 ± 0.2 16.87 ± 0.2 23.01 ± 0.1 20.44 ± 0.3 25.62 ± 0.3 22.98 ± 2.0

Table 4.4 Effect of seed inoculation of Enterobacter cloacae MSA1 on the vegetative growth of Jatropha curcas up to 120 DAS (days after sowing); result shows mean ± SE (standard error), C = Control; MSA1 = Enterobacter cloacae MSA1

28.32 ± 0.2 Fresh wt. (gm) 0.594 ± 0.4 1.131 ± 0.3 0.973 ± 0.2 2.32 ± 0.8 3.21 ± 0.4 4.87 ± 0.4 4.72 ± 0.8 9.74 ± 0.1

Root

Dry wt. (gm) 0.123 ± 0.04 0.213 ± 0.03 0.309 ± 0.05 0.532 ± 0.2 0.872 ± 0.07 0.932 ± 0.04 0.892 ± 0.02 1.886 ± 0.2 Length (cm) 13.29 ± 0.2 14.74 ± 0.3 15.55 ± 0.9 15.93 ± 1.4 19.34 ± 0.4 22.68 ± 1.3 20.23 ± 1.3 25.93 ± 2.8 Fresh wt. (gm) 4.33 ± 0.7 5.67 ± 0.8 8.54 ± 0.5 18.33 ± 1.6 10.92 ± 1.3 20.44 ± 1.2 11.23 ± 0.3 22.77 ± 1.2 Dry wt. (gm) 1.12 ± 0.3 2.13 ± 0.2 2.89 ± 0.6 5.90 ± 0.6 3.27 ± 0.7 6.01 ± 1.3 4.89 ± 0.4 8.01 ± 0.4

Shoot

Width (mm) 18.4 ± 1.2 23.1 ± 0.4 29.4 ± 2.2 34.7 ± 0.8 33.89 ± 0.4 36.86 ± 0.2 39.82 ± 0.32 48.65 ± 0.3 Number of leaf 4.66 ± 0.3 5.66 ± 0.3 4.33 ± 0.3 6.66 ± 0.3 5.33 ± 0.3 8.33 ± 0.3 8.33 ± 0.3 9.66 ± 0.3 Length (cm) 8.22 ± 0.4 9.74 ± 0.2 5.92 ± 0.9 9.83 ± 0.2 7.23 ± 0.5 10.12 ± 0.1 9.61 ± 0.7 12.22 ± 0.2

Width (cm) 7.26 ± 0.6 8.33 ± 0.2 6.82 ± 0.6 9.33 ± 0.3 7.46 ± 0.7 10.01 ± 0.2 9.87 ± 0.5 11.22 ± 0.1

Leaf Chlorophyll (mg/g)

0.731 ± 0.54 1.02 ± 0.32 0.93 ± 0.82 1.21 ± 0.42 1.01 ± 0.23 1.59 ± 0.7 1.21 ± 0.77 1.93 ± 1.2

Biomass (gm) 3.68 ± 0.9 4.45 ± 2.3 6.31 ± 0.4 14.21 ± 1.2 9.98 ± 0.8 18.36 ± 1.3 10.16 ± 0.7 22.61 ± 0.8

166


30 DAS 60 DAS 90 DAS 120 DAS Vegetative parameters C MSA2 C MSA2 C MSA2 C MSA2

Length (cm) 14.54 ± 0.3 18.23 ± 0.3 16.87 ± 0.2 22.12 ± 0.3 20.44 ± 0.3 23.89 ± 0.1 22.98 ± 2.0 27.84 ± 1.3 Fresh wt. (gm) 0.594 ± 0.4 0.973 ± 0.2 0.973 ± 0.2 1.43 ± 1.4 3.21 ± 0.4 4.29 ± 0.3 4.72 ± 0.8 8.53 ± 1.2

Root

Dry wt. (gm) 0.123 ± 0.04 0.212 ± 0.08 0.309 ± 0.05 0.693 ± 0.2 0.872 ± 0.07 0.897 ± 0.08 0.892 ± 0.02 1.826 ± 0.1 Length (cm) 13.29 ± 0.2 14.21 ± 0.3 15.55 ± 0.9 15.92 ± 1.3 19.34 ± 0.4 20.48 ± 1.2 20.23 ± 1.3 25.86 ± 2.4 Fresh wt. (gm) 4.33 ± 0.7 5.22 ± 1.2 8.54 ± 0.5 18.79 ± 1.2 10.92 ± 1.3 21.22 ± 0.4 11.23 ± 0.3 21.22 ± 0.6 Dry wt. (gm) 1.12 ± 0.3 2.07 ± 0.1 2.89 ± 0.6 5.94 ± 0.8 3.27 ± 0.7 6.03 ± 0.3 4.89 ± 0.4 7.16 ± 0.2

Shoot

Width (mm) 18.4 ± 1.2 22.3 ± 0.5 29.4 ± 2.2 33 ± 3.1 33.89 ± 0.4 34.66 ± 0.7 39.82 ± 0.32 43.65 ± 0.8 Number of leaf 4.66 ± 0.3 5.33 ± 0.3 4.33 ± 0.3 5.66 ± 0.3 5.33 ± 0.3 6.66 ± 0.3 8.33 ± 0.3 9.66 ± 0.3 Length (cm) 8.22 ± 0.4 9.33 ± 0.3 5.92 ± 0.9 6.73 ± 0.2 7.23 ± 0.5 9.12 ± 0.3 9.61 ± 0.7 12.23 ± 0.2 Width (cm) 7.26 ± 0.6 8.12 ± 0.4 6.82 ± 0.6 7.69 ± 0.2 7.46 ± 0.7 9.01 ± 0.1 9.87 ± 0.5 11.21 ± 0.1

Leaf Chlorophyll (mg/g)

0.731 ± 0.54 0.905 ± 0.43 0.93 ± 0.82 1.02 ± 0.97 1.01 ± 0.23 1.21 ± 0.63 1.21 ± 0.77 1.82 ± 0.85

Biomass (gm) 3.68 ± 0.9 3.91 ± 1.0 6.31 ± 0.4 13.58 ± 0.7 9.98 ± 0.8 18.58 ± 0.3 10.16 ± 0.7 19.02 ± 0.8 Table 4.5 Effect of seed inoculation of Enterobacter cancerogenus MSA2 on the vegetative growth of Jatropha curcas up to 120 DAS (days after sowing); result shows mean ± SE (standard error), C = Control; MSA2 = Enterobacter cancerogenus MSA2

167


Table 4.6 Significance test by using ANOVA software at 30 and 60 DAS; * Significant at 5% (ANOVA); ** Significant at 1% as compared to control (ANOVA); ns, non-significant as compared to control (ANOVA)

30 DAS

60 DAS

Cd at 1%

Cd at 5%

CV

Significance

Cd at 1%

CV

Significance Cd at 5%

Vegetative parameter

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

Length (cm)

7.213

3.563

0.354

0.432

3.546

4.687

*

ns

2.454

3.432

0.877

0.984

3.232

3.783

*

**

Fresh wt. (gm)

2.476

2.387

0.974

0.998

6.345

4.378

ns

ns

2.896

2.674

0.974

0.964

6.565

6.784

ns

ns

Root

Dry wt. (gm)

3.786

2.674

0.623

0.784

3.478

3.647

**

*

4.673

4.673

0.754

0.798

3.323

3.983

*

*

Length (cm)

3.642

3.647

0.689

0.834

7.784

4.897

ns

ns

3.647

4.758

0.487

0.776

6.565

5.453

ns

ns

Fresh wt. (gm)

2.547

3.468

0.956

0.756

4.783

4.893

ns

ns

5.687

2.644

0.989

0.894

4.783

4.893

**

ns

Dry wt. (gm)

5.743

2.748

0.976

0.465

5.785

3.787

**

ns

3.748

3.748

0.434

0.879

5.785

5.354

ns

ns

Shoot

Width (mm)

3.687

4.737

0.957

0.284

2.784

1.748

ns

ns

5.782

4.556

1.321 0.965

7.342

4.345

ns

ns

No. of Leaf

3.648

3.671

0.897

0.873

4.752

4.758

ns

ns

3.423

4.524

0.778

0.759

3.452

6.564

ns

ns

Length (cm)

4.672

2.783

0.983

0.783

3.098

4.268

ns

ns

5.354

3.674

0.476

0.783

4.365

5.983

ns

ns

Width (cm)

3.782

2.648

0.982

0.793

5.783

3.896

ns

ns

7.535

3.674

0.675

0.675

3.562

6.541

ns

ns

Leaf

Chlorophyll (mg/g)

3.783

4.782

0.492

0.389

4.892

7.893

*

ns

8.453

4.672

0.897

0.879

5.563

8.459

ns

ns

Biomass (gm)

7.893

2.784

3.676

2.534

8.354

7.363

*

*

3.525

4.673 2.564 0.933 12.543

10.645

**

**

168

169

90 DAS

120 DAS Cd at 1%

Cd at 5%

CV

Significance

Cd at 1%

Cd at 5%

CV

Significance

Vegetative parameter

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

MSA1

MSA2

Length (cm)

3.467

6.296

2.657

2.453

2.832

3.233

*

**

7.239

9.011

2.786

4.148

3.425

5.285

ns

ns

Fresh wt. (gm)

8.356

1.763

5.239

0.992

5.762

3.898

ns

*

4.963

0.813

0.184

0.374

4.538

2.190

ns

ns

Root

Dry wt. (gm)

2.656

1.564

0.452

0.983

4.879

4.362

ns

ns

2.481

1.094

0.327

0.504

2.634

15.814

*

ns

Length (cm)

4.856

2.374

0.784

0.342

2.983

4.673

*

ns

3.451

1.561

0.459

0.718

3.714

0.985

ns

*

Fresh wt. (gm)

7.634

9.646

2.563

1.782

5.632

4.785

ns

ns

10.673

7.845

4.825

3.611

3.969

6.310

*

**

Dry wt. (gm)

4.547

2.435

0.989

0.549

2.672

1.658

ns

ns

3.762

1.468

0.853

0.676

3.232

3.526

**

**

Shoot

Width (mm)

9.873

7.564

0.957

0.893

2.867

3.463

**

*

11.13

10.05

2.532

4.627

2.821

3.340

ns

ns

No. of Leaf

4.642

3.232

0.657

0.912

4.434

5.613

ns

ns

8.672

9.640

3.649

4.437

7.142

14.54

ns

ns

Length (cm)

2.643

3.278

0.453

0.872

5.623

5.765

ns

ns

2.648

1.577

0.958

0.726

5.621

2.186

ns

*

Width (cm)

1.897

4.732

0.343

0.784

4.862

7.232

ns

ns

2.482

1.667

0.968

0.767

3.562

2.113

*

*

Leaf

Chlorophyll (mg/g)

1.213

4.342

0.328

0.992

5.656

5.432

ns

ns

4.274

1.140

0.786

0.524

3.221

9.486

ns

ns

Biomass (gm)

13.878

10.978

12.132

9.731

7.674

8.374

*

*

14.672

18.99

6.758

8.742

12.547

17.976

**

ns

Table 4.7 Significance test by using ANOVA software at 90 and 120 DAS; * Significant at 5% (ANOVA); ** Significant at 1% as compared to control (ANOVA); ns, non-significant as compared to control (ANOVA)



Control MSA1

Picture 4.4 Comparative study of the vegetative structure of Jatropha with MSA1 90 DAS.

Control MSA2 Picture 4.5 Comparative study of the vegetative structure of Jatropha with MSA2 90 DAS.

170


Jatropha curcas compares to control 90 DAS (in

MSA1 MSA1 MSA2 MSA2 MSA1 MSA2 Control

Picture 4.6 Green house study of

triplicate)

171


172

Inoculation of Jatropha seeds with ACC-deaminase containing rhizobacterial isolates

significantly increased root elongation in both trials. The root length of Jatropha curcas

seedlings was significantly increased by inoculation with MSA2 that shows 25.38, 31.12,

16.88 and 124.14 % from 30, 60, 90 and 120 DAS over uninoculated control. Similarly

caused increase in the fresh root weight of Jatropha seedlings over uninoculated control

that ranged from 34 to 81 %, while dry weight shows the increase from 2.8 to 124 %.

Data revealed that shoot length of Jatropha seeds was significantly increased due to

inoculation with ACC-deaminase containing rhizobacteria from 6.99, 2.37, 5.89 to 27.83

% within 30, 60, 90 and 120 DAS, similarly fresh shoot weight increased 20.55 to 120.02

% and the dry shoot weight increased 46.42 to 105.54 % from 30 to 120 DAS

respectively. Shoot width of Jatropha was calculated and found that 2.2 to 21.20 %

increased over uninoculated control from 30 to 120 DAS.

Isolate MSA2 was also effective in significantly increasing the number of leaves per

plant of Jatropha resulted in to 14.38, 30.72, 24.45 and 15.96 from 30,60, 90, and 120

DAS compared with their respective uninoculated control. While the leaf length and leaf

width increased 13.50 to 27.26 % and 11.85 to 20.77 % from 30 to 120 DAS. Total

chlorophyll content increased 23.80, 9.68, 19.80 and 50.41 % from 30, 60, 90 and 120

DAS over the uninoculated control. Biomass of Jatropha curcas was also increased over

control after inoculation with the culture MSA2 and resulted into 6.25, 115.21, 86.17 and

87.20 % from 30, 60, 90 and 120 DAS. The data were subjected to analysis of variance

(ANOVA) with their mean values of three replicates (Table 4.6 and 4.7) in each

treatment were compared using least significant differences at 5% probability (P=0.05).

Seed bacterization has proved a successful method for enhancing biological control of

plant diseases. In this study, plants treated with the MSA2 isolate showed stimulatory

effects on all plant vegetative parameters. It is highly likely that the ability of these ACC

enriched rhizobacterial isolates to deaminate ACC was the responsible mechanism of

action for promoted root and shoot growth because lowering of the ACC levels result in

decreased endogenous ethylene production. This contention is strongly supported by the

work reported by several other researchers (Glick et al. 1998; Mayak et al. 1999).


173

The variation in growth promotion by different isolates may be due to the differences in

their efficiency to colonize the germinating roots and deaminating the ACC formed in

roots. The isolates with greater efficiency of deaminating endogenous ACC might have

caused more root growth promotion by eliminating the inhibitory effects of higher

ethylene concentrations produced from endogenous ACC. Similar results have been

reported by others (Shah et al. 1998; Li et al. 2000; Wang et al. 2000). Moreover, the

rhizobacterial isolates might have produced other biologically active substances, which

had affected the growth. Isolates Enterobacter cloacae MSA1 and Enterobacter

cancerogenus MSA2 were found to be the most effective in increasing root length, shoot

length, and fresh weight as compared with other control selected for experiments. This

positive effect of inoculation could be due to a decrease in ethylene synthesis in

inoculated roots. Many researchers have reported that under gnotobiotic conditions, seed

and/or root inoculation with rhizobacteria promotes root growth through ACC deaminase

activity (Glick et al. 1998). It was found that the bacterial strain without ACC deaminase

did not promote the growth of inoculated plants, confirming that the ACC deaminase trait

of PGPR was primarily responsible for growth promotion.

The most consistent effect of Enterobacter cloacae MSA1 was observed as increased

root fresh weight and dry weight ie 138.43% and 111.43 % while the root length

increased up to 36.96% it shown that inoculation of MSA1 enhances more number of

root branching instesd of length. Shoot fresh and dry weight increases up to 114.63% and

525.07% was consistent and much higher compared to control. Chlorophyll content also

increases continuously up to 59.50%. Enterobacter cancerogenes MSA2 was observed

as increasing root length and root dry weight, which increased up to 124 % and 104 %

four months days after inoculation. Similar kind of report available with the growth

promotion of Jatropha curcas by the isolate Bacillus pumilis (IM-3) increases shoot

length up to 113 % and the total chlorophyll content up to 82 % . (Desai et al. 2007).


174

4.6 Conclusion

Undoubtedly, the isolation of Enterobacter cloacae MSA1 and Enterobacter

cancerogenus MSA2 as their very attractive features for agronomic applications, some

detected for the first time in these species, emphasizes the significance of performing

studies on suitability of exploring common environments, such as the rhizosphere, for

isolation of bacterial species with biotechnological potential. Finally, preliminary

indications are that ACC deaminase-containing PGPR strains are beneficial in the field as

well as under more controlled laboratory conditions. We isolates these two isolates have

been more beneficial in the field conditions as well with the jatropha plants in the land of

Gujarat, India. It is envisioned that these isolates will help to ameliorate some of the

deleterious effects of a variety of environmental stresses on plants. Isolates MSA1 and

MSA2 were found the suitable candidate for the pot and field study with the different

leguminous and non leguminous plants as they show good plant growth promoting

potentials. The ability of Enterobacter cloacae and Enterobacter cancerogenus to

produce ACC deaminase, together with its inherent mechanisms to promote plant growth,

may render this bacterium very useful in an agricultural setting. There is considerable

experimental evidence that certain microorganisms are able to colonize the root–soil

environments where they carry out a variety of interactive activities known to benefit

plant growth and health, and also soil quality. Given the current reluctance of many

consumers worldwide to embrace the use as foods of genetically modified plants, it may

be advantageous to use plant growth-promoting bacteria as a means to promote growth

by lowering plant ethylene levels or reduce disease through induction of resistance, rather

than genetically modifying the plant itself to the same end.

Rhizobacteria having ACC deaminase activity are effective in promoting plant growth

and water use efficiency under drought conditions, by lowering the ethylene or ACC

accumulation whose higher levels have inhibitory effects on root and shoot growth.


175

From the previous demonstrations, it is established that the microorganisms that possess

ACC deaminase activity have the selective advantage over other bacteria during biotic

and abiotic stress conditions. Besides the activity of ACC deaminase in alleviating

ethylene- mediated abiotic and biotic stresses, the ecology of bacterium and physiology

of the plant may also interact with plant system to increase resistance to stress. However,

the defined mechanisms involved in the use of plant growth-promoting rhizobacteria

which decrease the damage to plants that occurs under stress conditions is a potentially

important adjuvant to agricultural practice in locales where stress is a major constraint.

ACC deaminase producing rhizobacteria often possess biocontrol potentials by producing

HCN (hydrocyanic acid), and some other enzymes like cellulose, chitinase and β-1, 3

glucanase against the fungal pathogens. Using transgenic approaches like horizontal

transfer of ACD (ACC deaminase) genes, the action of bacterial ACD on plant

development have been studied in a wide variety of models. It has been shown to delay

fruit ripening, increase tolerance to heavy metals and decrease susceptibility to a variety

of environmental stresses. During last decade, understanding about the plant growth

promoting rhizobacteria producing ACC deaminases has increased upto their molecular

level leading to a number of commercial applications.

Documents

Characterization of ACC deaminase - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4342/14/14_chapter 4.pdf · Characterization of ACC deaminase 125 walls or nematode structures