Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC Deaminase Activity on Root Length of <i>Lens esculenta</i> Seedlings

doi:10.4236/ajps.2013.411273

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American Journal of Plant Sciences, 2013, 4, 2199-2205

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411273

Open Access AJPS

2199

Effect of Inoculation with Three Phytohormone Producers

Phytobacteria with ACC Deaminase Activity on Root

Length of Lens esculenta Seedlings

Natalia Elenes Zazueta1, Orlando Ortega Acosta2, Laura Martínez Herrera2, Raúl Alcalde Vázquez2,

Eugenia López López2, Angelica Guerrero Zúñiga3, Angelica Rodríguez Dorantes2*

1Advanced Technological Institute of Cajeme, Sonora, México; 2National School of Biological Sciences, I.P.N., México City,

México; 3Mexican Petroleum Institute, México City, México.

Email: *[email protected]

Received September 21st, 2013; revised October 20th, 2013; revised October 27th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Plant-associated bacteria that inhabit the rhizosphere may influence the plant growth by their contribution to the en-

dogenous pool of phytohormones and by the activity of ACC deaminase to decrease the ethylene concentration. The

aim of this study was to analyse the root length growth by the promoting effect of indole acetic acid producers phyto-

bacteria with ACC deaminase activity, on inoculated seeds of Lens esculenta as synergistic effect on root elongation. In

this study, although the roots of L. esculenta seedlings do not show a significant promotion, these phytobacteria could

be recommended to treat plants analyzing their added inoculum to increase plant biomass and retard the effect of ethyl-

ene on cultures supplied with Tryptophan and ACC.

Keywords: Plant Growth-Promoting Bacteria; Lens esculenta; Root Elongation Test; Indole Acetic Acid; ACC

Deaminase Activity

1. Introduction

The 1-aminocyclopropane-1-carboxylate (ACC) deami-

nase (ACC deaminase) hydrolyses the 1-aminocyclo-

propane-1-carboxylate (ACC) into ammonia and α-ke-

tobutyrate instead of its conversion into ethylene; the

uptake and cleavage of ACC by this enzyme decrease its

amount and as consequence the ethylene concentration in

plants [1-5]. Authors reported that the ACC deaminase

trait has been extensively studied in numerous soil mi-

croorganisms, but it is most common among plant

growth promoting rhizobacteria to protect plants from

both biotic and abiotic stresses and favor the increase of

plant biomass through the regulation of ethylene synthe-

sis in inoculated plants [5-14]. Bacteria that inhabit the

rhizosphere also may influence the plant growth by their

contribution to the endogenous pool of phytohormones,

such as auxins in plants; the production of the auxin Indole

Acetic Acid (IAA) is reported among plant-associated

bacteria [15]. The aim of this study was to analyze the

root length growth by the promoting effect of indole ace-

tic acid producers phytobacteria with ACC deaminase

activity on inoculated seeds of Lens esculenta as syner-

gistic effect on root elongation.

2. Materials and Methods

2.1. Evaluation of the IAA Production of the

Selected Phytobacteria

The phytobacteria employed: Lemna 2 strain, U-M1-4

strain and U-M1-5 strain, were isolated from the aquatic

plant Lemna gibba, collected from the Lake Xochimilco,

México. The selected phytobacteria were analyzed by

their Indole Acetic Acid (IAA) production [16,17] using

the Salkowski reagent according to the method of Bric et

al. [18] and Melo et al. [19], taking 4.9 mL of sterile

Luria-Bertani (LB) liquid media, added to culture tubes

(10 × 15 cm); the culture tubes were inoculated with 0.1

mL of each phytobacteria with an adjusted inoculum by

optical density of 5 × 107 cells/mL in sterile distilled wa-

ter and incubated at 28˚C for 120 h. After the incubation,

*Corresponding author.

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC

Deaminase Activity on Root Length of Lens esculenta Seedlings

2200

the cultures were centrifuged at 3,500 rpm, at 25˚C for

45 minutes to discard the bacteria pellets, the supernatant

was recovered and 2 mL of each supernatants were

mixed with 2 mL of Salkowski’s coloring reagent, the

development of a pink color indicates IAA production

and was quantified reading its absorbance at 535 nm. The

concentration of IAA was estimated by a standard curve

and the assays were performed by triplicate.

Evaluation of the ACC deaminase activity of the se-

lected phytobacteria The ACC deaminase activity of the

isolates was assayed according to the method of Penrose

and Glick [20] and Khandelwal and Sindhu [21], with

ACC and (NH4)2SO4 as sole nitrogen source. The assay

was done using agar plates with DF minimal medium [22]

supplemented with ACC (1mM) or (NH4)2SO4 (2 g/L),

equally divided into 24 sectors and spot inoculated with a

sterile toothpick each colony of the selected phytobacte-

ria. The plates were incubated at 28˚C for 48 to 72 h and

the presence of ACC deaminase activity, was recorded

by the measurement of the diameter of the colonies and

compared between the two nitrogen conditions. The as-

says were performed by duplicate.

2.2. Effect on Root Growth of Lens esculenta

Seedlings Inoculated with the Selected IAA

Producers with ACC Deaminase Activity

Phytobacteria

Bacterial inoculum were obtained by culturing the

phytobacteria strains on plates with LB agar medium for

48 h at 28˚C and re-suspending in sterile distilled water

to adjust by optical density an inoculum with cell density

of 5 × 107 cells/mL; 0.1 mL of the bacterial suspensions

were spread on Petri dishes containing mineral medium

with phytagel, added with Tryptophan (2 mg/L) and/or

ACC (1 mM). The plates were stand for 30 minutes for

the inoculum absorption. Twenty five commercially

seeds of Lens esculenta were surface-sterilized with 10%

sodium hypochlorite and then thoroughly rinsed with

sterile distilled water and placed in the Petri dishes, with

the respective conditions. Petri dishes with mineral

medium containing: 0.20 M NH4H2PO4, 0.50 M NH4NO3,

1.15 M Ca(NO3)2, 0.26 M CaCl2, 0.2 M MgCl2·6H2O,

0.20 M Mg(NO3)2·6H2O, 0.40 M MgSO4·7H2O, 0.20 M

KH2PO4, 1.2 M KNO3, 0.5 M K2SO4, 0.04 M

FeCl3·6H2O, 1.2 × 10−2 M H3BO3, 1.2 × 10−4 M

CuCl2·H2O, 2.3 × 10−3 M ZnCl2, 4.4 × 10−4 M

MnCl2·4H2O, 6 × 10−6 M Na2MoO 4·H2O, EDTA and

FeSO4·7H2O, pH = ±6.0, and 3 g of phytagel; un-

inoculated treatments were considered as blanks. All the

experiments were performed by duplicate and main-

tained at 30 °C in a growth chamber in dark for 4 days.

The plant root elongation promoting (PREP) activity

assay was employed to analyze the promoting and

synergistic effect of rhizobacteria strains on Lens esculenta

seeds, according to the modified root elongation assay of

Belimov et al. [23], the root length of the seedlings were

measure and the Growth Index (GI), expressed as the

ratio of the root lengths of plants grown in the presence

and absence of the phytobacteria, was obtained [14]; GI

= RLpb/RLc, where RLpb is the root length of plants

grown in the presence of the specific phytobacteria

inoculum and RLc is the root length of plants grown in

absence of inoculum (control).

2.3. Statistical Analysis

All data were analysed by One-way ANOVA analysis of

variance and the mean differences were compared ap-

plying a Tukey-Kramer post-test, using the statistics

program Graph Pad Instat Ver. 3.10.

3. Results

3.1. IAA Production and ACC Deaminase

Activity by the Tested Strains

Lemna 2 strain and U-M1-4 strain presented a higher

ACC deaminase activity (p < 0.001) than U-M1-5 strain,

according to their colonial diameter (Figure 1). The em-

ploy in the metabolic assay of ammonium sulfate as ni-

trogen source, was only taking it as control, compared to

the ACC assay, but in this study, the two phytobacteria

mentioned had a minor colony diameter, compared to the

ACC activity. The three phytobacteria presented an in-

crease in the IAA production as the tryptophan supply in

cultures increased, the basal production of this auxin

without the aminoacid presented this order: U-M1-5

strain (32.67 μg/mL) > U-M1-4 strain (30.7 μg/mL) >

Lemna 2 strain (19.32 μg/mL), all the bacterial isolates

were classified according to Khalid et al. [24] as higher

phytohormone producers (Figure 2).

3.2. Root Elongation Test of L. esculenta and the

Effect of Phytobacteria Inoculation

The response of L. esculenta roots showed that this spe-

cies was susceptible to the presence of Trp, ACC and Trp

+ ACC, with a significant reduction on the root devel-

opment compared to the roots grown on mineral medium:

U-M1-4 strain 55% > U-M1-5 strain 36.17% > Lemna 2

strain 27.95% (p < 0.001). In general, inoculation with

the three phytobacteria strains decrease the root length

compared to the control roots (U-M1-4 strain > U-M1-5

strain > Lemna 2 strain). Particularly, the effect of the

seeds inoculated with the strains and Trp showed that the

order of response was as follows: U-M1-5 strain >

U-M1-4 strain > Lemna 2 strain), the inoculated seeds

and ACC: Lemna 2 strain > UM1-5 strain > UM1-4 and

Open Access AJPS

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC

Deaminase Activity on Root Length of Lens esculenta Seedlings

Open Access AJPS

2201

Figure 1. ACC deaminase activity of the phytobacteria tested: (A) DF minimal medium with ACC and (B) DF minimal me-

dium with Ammonium Sulfate, where: “a and b” Lemna 2 strain, “c and d” U-M1-5 strain, “e and f” U-M1-6 strain. Mean

values ± S.D. from 48 replicates.

the inoculated seeds and Trp + ACC: UM1-5 strain >

Lemna 2 strain > U-M1-4 strain (Figures 3 and 4).

measured. It is known from application of exogenous

IAA [26] or application of diluted culture extracts or low

density inoculum of bacteria that produce high levels of

IAA [27,28] that low concentrations of IAA can stimu-

late primary root elongation; it is important to note that the

effect of bacterial IAA on plant growth depends on the

size of the inoculum of a single strain, but not always,

4. Discussion

Glick et al. [25] showed that the promotion of root

growth is one of the principal markers by which the

beneficial effect of plant growth-promoting bacteria is

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC

Deaminase Activity on Root Length of Lens esculenta Seedlings

2202

Figure 2. IAA in vitro production of the phytobacteria

tested. Mean values ± S.D. from three replicates.

the inoculum density employed means that more IAA is

available to the plant, and reports of bacterial mutants that

overproduce IAA show a root growth-inhibiting effect

[29-31]. Burd et al. [14] and Belimov et al. [32] reported

that there is a number of plant growth promoting bacteria

that contain the enzyme ACC deaminase and stimulate

the root growth of different plant species; ACC is exuded

from roots or seeds and cleaved by ACC deaminase to

NH3 and a-ketobutyrate, the bacteria utilize the NH3 as a

source of nitrogen and thereby decrease ACC within the

plant [9,33] with the concomitant reduction of plant eth-

ylene [4,14,34,35]. Patten and Glick [36] demonstrated

that the IAA secreted by a bacterium may promote root

growth directly by stimulating plant cell elongation or

cell division or indirectly by influencing bacterial ACC

deaminase activity. The authors mention that the IAA

and ACC deaminase work together to stimulate the root

elongation; this consideration is regarding to the exoge-

nous IAA that increase the transcription and activity of

ACC synthase; this enzyme catalyzes the production of

ACC in plants and therefore ACC stimulates the ACC

deaminase activity in bacteria [6,37], while the IAA

produced by the bacterial inoculum, stimulates the root

elongation or the formation of lateral and adventitious

roots [29-31,38]. In this study, the correlation between

the IAA production and the ACC deaminase activity by

the phytobacteria tested, showed a relationship between

the U-M1-4 and U-M1-5 strains that were joined together

as one group by this attribute, separated to the Lemna 2

strain; probably the density of the inoculum produced a

high IAA concentration that generated a inhibiting effect

on root growth, joined to the ACC metabolism activity

presented by the phytobacteria. This attribution is related

with the visible short and thick appearance of the roots

showed in the treatments with ACC and Trp + ACC. The

effect of the inoculation with these three phytobacteria

strains on Lens esculenta seeds, doesn’t show an evident

increase in the root length of seedlings; but particularly

was the response of two of the isolates: Lemna 2 strain and

Figure 3. Root length measurement of Lens esculenta seed-

lings: (a) Control experiments, (b) Trp, ACC and Trp +

ACC inoculated with Lemna 2 strain, (c) Trp, ACC and Trp

+ ACC inoculated with U-M1-4 strain and (d) Trp, ACC and

Trp + ACC inoculated with U-M1-5 strain. Mean values ±

S.D. from 50 replicates, the different bold letters show the

significant differences between experiments (p < 0.001).

Open Access AJPS

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC

Deaminase Activity on Root Length of Lens esculenta Seedlings

2203

Figure 4. Root length experiments of Lens esculenta seeds

inoculated with U-M1-5 strain: (a) Mineral Medium, (b)

Mineral Medium + ACC, (c) Mineral Medium + Trp and (d)

Mineral Medium + Trp + ACC.

U-M1-5 strain with a relationship according to their

measured attributes: Lemna 2 strain with a high ACC

activity (with a 47.32% of root growth compared to the

control roots) and U-M1-5 strain with the highest in vitro

IAA production (with a 94.98% of root growth compared

to the control roots) especially with the presence of Trp

(2 mg/L). Even the root length of the seedlings was lesser

than control seedlings for both strains; the seeds’ treat-

ment with Trp + ACC showed that the response of the

root length was higher in the experiments with TRP with

U-M1-5 strain (with a 78.26% of root growth compared

to the control roots) than Lemna 2 strain (with a 71.09%

of root growth compared to the control roots).

These results were according to the results obtained by

Zafar-Ul-Hye et al. [39]; these authors screened rhizo-

bacteria containing ACC deaminase to promote lentil

growth under axenic conditions and by Shaharoona et al.

[5], who analyzed different strains of rhizobacteria with

variations in their ACC deaminase activity, capability of

the rhizobacterial isolates of IAA production in the pres-

ence and absence of tryptophane and isolates also varied

in their ability to colonize etiolated pea roots; both ac-

cording to the reports by Shaharoona et al. [40] and

Zafar-Ul-Hye et al. [39] where the bacteria with more

ACC deaminase activity had more ability to decrease the

intensity of the called “ACC-induced classical triple re-

sponse”, the ACC deaminase activity of bacteria was

responsible for the decrease of endogenous and exoge-

nous ACC supply in inoculated plant with the inhibition

on the root length of L. esculenta seedlings (Lemna 2

strain to the control roots). Finally, although the roots of

L. esculenta seedlings do not show a significant promo-

tion with the presence of the strains, Trp and ACC in

medium; and even though these responses were adverse,

the results obtained in this work suggested a synergistic

effect between the two bacterial attributes probed and the

phytobacteria tested and could recommended that it is

important to consider the inoculum density to the in-

crease of plant biomass and retard the effect of ethylene

on cultures supplied with Tryptophan and ACC.

5. Acknowledgements

Authors are grateful to the Research Projects: SIP:

20131494 of the Secretaría de Investigación y Posgrado

del I.P.N. and ISITDF/325/11 AREAS PRIORITARIAS-

IPN and COFAA-IPN, EDI-IPN, SNI-CONACYT fel-

lowships.

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Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC

Deaminase Activity on Root Length of Lens esculenta Seedlings

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