Identification and quantification of IAA and IBA in Azospirillum brasilense-inoculated maize roots

Soil Bid B&hem. Val. 21, No. 1. pp. 147-133. I989 Printed in Great Britain. All tights reserved

0038-0717l%9 $3.00 + 0.00 Copyright ,C 1989 Pcrgamon Press pk

IDENTIFICATION AND QUANTIFICATION OF IAA AND IBA IN ~Z~SPr~~~~U~ ~~~SrLE~~~-INOCULATED MAIZE ROOTS ELAZARFALIX and The

YAACOV

CkoN

Hebrew University of Jerusalem. Faculty of Agriculture, Department of Plant Pathology and Microbiology, P.O. Box 12, Rehovot 76tO0, Israel

EPHRAIMEPSTEIN Department of Horticulture. Agricultural Research Organization The Volcani Center, Bet-Dagan 50250, Israel

ALEXANDER GOLDMAN* The Hebrew University of Jerusalem, Faculty of Agriculture. Department of Biochemistry, P.D. Box 12, Rehovot 76100, Israel

and MEIR FISCHER Biot~h~ology General (Israel) Ltd. Kiriat Weizmann, Rehovot 76326, Israel (Accepted 27 June 1988) Summary-fnoculation of maize seedlings with IO’ colony forming units (cfu) of Axwpirillum plant-’ significantly increased the root surface area 2 weeks after sowing as compared to non-inoculated plants. Indole-3-acetic acid (IAA) was identified by gas liquid chromatography (GLC) and gas chromatography-rna~ spectrometry (GC-MS) in ~~o~~~r~~~urn tryptuphan-fry culture medium. The amount of IAA in the medium was calculated From the gas chromatogram by isotope dilution analysis and found to be 32-4Ong ml-‘. Roots of A=ospirillrcm-inoculatedmaize seedlings were found to have higher amounts of both free and bound IAA as compared to control. The amount of free IAA signi~cantly increased in the inoculated roots 2 weeks after sowing. IAA and indole-3-butyric acid (IBA) were identified by GC-MS only in the roots of A,-ospiriNum-inoculated seedlings 2 weeks after sowing. The significance of the higher amounts of IAA following inoculation with the bacteria is discussed.

Investigation

of rhizosphere

bacteria

0.1 PM-IO PM induces root elongation, ceil division in meristemic tissues, cell differentiation and the formation of adventitious roots. Higher concentrations of IAA, I mM--1PM, in the root cause inhibition of growth (Elliott, 1982). About 95--98% of the IAA in maize seed is found as an ester conjugate of IAA (Epstein et al., 1980). During seed germination, the IAA-conjugates in the endosperm are metabolized in the vascular tissue of the plant by enzymes (Nonhebel et al., 1985) to give active free IAA (Nowacki and Bandurski, 1980). There is no evidence to show that Azos~ir~~~~rn affects endogenous levels of IAA in roots of grasses. The purposes of this work were: (1) to quantify and identify the amount of IAA in Azospiriii~m brusiiense-Cd in a tryptophan-free growth medium; (2) to detect differences in conjugated and free IAA content in A~ospi~i~lum inoculated and noninoculated roots; and (3) to compare IAA metabolism in the inoculated vs non-inoculated roots.

of the genus

Axupirillum

during the past decade has indicated that these bacteria benefit forage and grain grasses under various environmental and soil conditions (Kapulnik et at., 1983; Reynders and Vlassak, 1982; Sarig et al., 1988). At a concentration of lo’--10’ Azospirifiwn plant-‘, the bacteria increased root hair development, root branching and root surface area (Fallik er al., 1988; Okon and Kapulnik, 1986), improved water status of the plant (Sarig et ai., 1988) and increased the yield of grasses between l&15% (Okon, 1985). A concentration of IO*-IO9A:osp~fi~~~m plant-i inhibited plant growth (Okon and Kapulnik, 1986). In culture, Axwpirillum is capable of producing hormone-like substances including IAA (Hartmann ef a/., 1983; Horemans and Vfassak, 1985). It has been suggested that Arospirilhm affects root growth and function by producing plant growth regulators (Inbal and Feldman, 1982; Kolb and Martin, 1985; Morgenstern and Okon. 1987). Indole-3-acetic acid (IAA) at concentrations of

iMATERIALS

AND METHODS

Biological material A~vspiri~~umbrsi/ense ATCC 29729 (Cd) was used

*Deceased.

in all experiments. 147

The growth conditions

of the

ELAZAR

FALLIKetat.

I 5

M

Fig. t. Capillary gas chromatographic elution profile of an authentic of PFT-IAA (A) and IAA sample from Axspirillunr culture medium (B). Fig. I. Root surface area of maize inoculated with IO’ cfu plant-’ (0) compared to non-inoculated plants (0). during 4 weeks from sowing (ml NaOH 0.1 M plant-‘). Points represent the means of 16 replications F SE at a probability of P = 0.05 using Duncan’s multiple range kospirilhm

test. bacteria, seed variety, seed growth and inoculation and pot experiments were described by Fallik et al. (1988). The concentration of Arospirilfum inoculant applied at sowing was 10’ cfu plant-‘. The total surface area of the root system was determined by the titration methods of Carely and Watson (1966). This method has proved to be the most reliable criterion in evaluation and measurement of growth response of maize to inoculation with A~ospirilfum (Fallik ef al., 1988). The plants which were used for the identification of indole metabolites by GC-MS, were grown for 2 weeks in steriIe sea sand and were watered with sterile tap water.

Determination

1s 3

too

(a)

so-

L-

Fig.

of IAA

production

in culture medium

was grown in 600 ml medium for 72 h (Fallik et a!., 1988) but without tryptophan or yeast extract. The number of bacteria after 72 h was 2 x IO’cfu ml-‘. The growth medium was centrifuged twice at 15,000 rev min-’ for 10 min and 20 ~1 of 4 x IO’dpm of [2-“C]IAA (2.18 x IO6Bq. Amersham Corporation, England) was added to the supernatant. The sample was brought to pIi 2.5 with 6~1 I-ICI and passed through a C,, Sep-Pak (Waters Associates) cartridge, twice. The cartridge was washed with distilled water and the IAA was eluted with 20 ml of distilled methanol. The methanol was evaporated under vacuum and the residue was taken up with 0.5 ml of distilled methanol:acetonitrile (1: I) mixture and transferred to reaction vials for derivatization. Derivatization, thin layer chromatography (TLC), GLC and GC-MS were performed as described for plant endogenous IAA. A~ospirillum

3(a)

IAA and IBA in inoculated maize roots

149

II-

(b)

)-

109 I

I

I

130

lnoculat~d rams,. Iscon c 16671

I A-Z

(cl

109

77

103 63

75

I

III

I 60

I 60

II, I 100

417 I,,

,1:3,

I 120

1::

,

1,

’ 140

160

160

M/Z Fig. 3. Mass spectra of authentic [AA-methyl ester (a), methylated IAA isolated from 72 h A:ospirihn culture medium (b) and methylated IAA isolated from extracts of 2-week old inoculated roots of maize seedlings (c). Determination seedlings

of endogcnoas

lAA

in roots

of maize

Samples of roots (IO g) were extracted in 70% cold acetone by a Waring blender (Bandurski and Schulze, 1977). After homogenizing, 4 x IO’dpm of (2-“C]IAA (2.18 x IO6Bq, Amersham Corporation, England) was added to each sample. Further extraction and purification procedures using DEAESephadex, Sep-Pak cartridge, and thin layer chromatography (TLC) are described by Epstein et al. (1986). The purified samples were fluorinated with z-bromo-2,3,4.5.6. pentafluorotoluene (PFT) (Aldrich) according to Epstein and Cohen (1981) for analysis in GLC. GLC was performed using a Varian 3300 equipped with a 6’Ni electron capture detector

(ECD) (Epstein and Cohen, 1981). NJ was used as a carrier gas at I ml min-’ with a 30 m Durabond-5 capillary column (J&W, Scientific Company, Folsom, Calif.). Injections were made in the splitless mode and methanol was used as the injection solvent. The temperatures of the column, the detector and the injection chamber were 220, 320 and 28OC. respectively. Under these conditions IAA-PFT had a retention time of 13.5 min. For standardization of detector response, a sample of [2-‘JC]IAA was fluorinated as above and eluted from the TLC plate. The amount of the IAA in I ~1 was calcualted from its radioactivity to be 200 pg PI-‘. This was then used to calculate the amount of IAA per unit area in the GLC peaks. Following the determination of radioactivity in each sample the

150

ELAZNI FALLIKeral.

amount of IAA in the tissue was calculated using the isotope dilution equation (Rittenberg and Foster, 1940). Gas chromatography-mass

spectrometry

Mass spectra were obtained on Finnigan-4610 GC-MS equipped with a SE-54 30 m column (J&W Scientific Company, Folsom, Calif.), 70 eV and column pressure of 10 psi. The temperature of the oven and the injection cell were 250 and 24O’C. respectively. The initial oven temperature was IOO’C programmed at 4°C min-’ up to 240°C and for another 5 min at 240°C. Identl$cation of IAA metabolites Fourteen-day-old maize seedlings were carefully removed from the pots, washed and transferred to a black container containing 50ml of sterilized tap water plus 100 ~1 of [2-14C]IAA. After 24 h of exposure, 20 g of roots were homogenized with an Ultra Turrax in 70% cold acetone and the homogenate was filtered. The filtrate was reduced in a vacuum to 4 ml. brought to pH 7.0 and loaded onto a 20 x 1.5 cm DEAE-Sephadex column. The column was eluted with 50ml of 50% isopropanol (neutral fraction), followed by a linear gradient elution with 125 ml of 50% isopropanol in H,O (v/v) and 125 ml of 50% isopropanol in Hz0 (v/v) pH 1.5 acidified with 3 ml phosphoric acid. Five ml were collected in each tube and radioactive fractions [acidic fraction (A)] were pooled for further purification. The acidic fraction was neutralized, dried under vacuum, dissolved in I ml distilled methanol and evaporated under N1 to 200 rtl for high-performanceliquid-chromatography (HPLC). HPLC analysis was performed on a Varian-5000 chromatograph with a Rehodyne 7125 sample injector (100 ~1 loop). a Hewlett-Packard 3390A reporting integrator and an analytical column (4.6 x I25 mm packed with Whatman ODS-85 reverse phase 5u column). The solvents were I% acetic acid in HZ0 (A) and 100% methanol (B). The solvent programme was O-30 min linear gradient from IO to 30% B and 3045 min linear gradient from 30 to 100% B, at a flow rate of 1 ml min-’ The eluant was monitored with a Varian UV-100 variable wavelength detector set at 280 nm and I ml fractions were collected using a Gilson 201 B fraction collector. Radioactive fractions were pooled and dried under vacuum and dissolved in distilled methanol to 200~1. Four distinct peaks were eluted and designated A, , A?, A, and A,. The four fractions were methylated with 1 ml of diazomethane in ether (Cohen, 1984) and were injected onto GC-MS for identification.

Oh-i-

k++4

WEEKS

FROM

SOWING

Fig. 4. Amount of conjugated (A) and free-IAA (B) (ng IAA g-’ fresh wt) in extracts of maize roots inoculated with IO’ Azospirillum plant-’ (0) compared to non-inoculated control (0). Points represent the means of 6 replications + SE at a probability of P = 0.05 using Duncan’s multiple range test.

Determination of IAA in the culture medium of Azospirillum The identification of IAA by GLC and GC-MS is shown in Figs 2 and 3. The PFT derivative of the putative IAA from the bacterial medium is represented in the graph by a peak at Rr = 13.5 min (Fig. 2). This peak is identical to the RT value of authentic

REStiLTS

Determination of the effect of inoculation with Arospirillum on root surface area Relative root surface area was determined in roots inoculated with 10’ cfu A:ospirillum plant-’ and non-inoculated controls, once a week for 4 weeks after sowing (Fig. I). Relative root surface area of inoculated plants was significantly higher the . . . after . second week compared to non-inoculated plants.

Fig. 5. For identification of IAA metabolites. ‘-weeks old inoculated and non-inoculated maize seedlings were exposed for 24 h to [2-“C]IAA and then roots were extracted. Partially purified extracts samples were eluted in HPLC and tested for their radioactivity. HPLC elution of (A) inocu-

-

lated root extracts and (B) non-maculated controls.

IAA

and IBA

in inoculated

The RT of the bacterial IAA sample was 13.5 + 0.1 min for replications. The amount of IAA in the culture medium of Azospirillum was calculated from its peak in GLC by the use of isotope dilution analysis and found to be 32AOng ml-‘. The mass spectrum of the extract yielded the fragmentation pattern typical of indoles and the molecular ion (M/Z = 189) of methyl ester of IAA (Fig. 3(b)]. IAA.

Determination of endogenous conjugated andfiee IAA in roots of maize seedlings The amount of conjugated and free IAA was determined in extracts of inoculated and noninoculated maize roots once a week for 4 weeks after sowing (Fig. 4). The amount of conjugated IAA in both inoculated and non-inoculated maize roots decreased during plant growth. The amount of conju-

maize roots

gated IAA in the inoculated roots was higher relative to the control and significant only during the fourth week after sowing [Fig. 4(A)]. The amount of free IAA [Fig. 4(B)] was significantly higher only after 2 weeks from sowing in inoculated roots. Identification of IAA metabolites in inoculated and control roots by W-MS HPLC of the acidic fraction from inoculated roots extract showed four distinct peaks which coincided with the radioactive peaks (Fig. S(A)], while that from the non-inoculated roots gave only one large peak at the beginning of the chromatogram [Fig. S(B)]. Elution time of fraction A, from the inoculated roots corresponded to that of authentic IAA (data not shown). GC-MS of fractions A, and A, (see Fig. 5) did not reveal any indole metabolites. IAA was identified in IEA-hfhyl

loo

151

w1.r

ISTO

(a)

50

217.q

;

9

100

-

(b)

217.1 143.1

166.1 156.2 d

Fig. 6. Mass spectra of authentic IBA-methyl old inoculated

140

160

100

200

220

ester (a) and methyl ester of IBA isolated extracts of 2-week roots of maize seedlings (b).

ELAZAR FALLIK ef al.

152

fraction Az (molecular ion of methyl ester of IAA, M/Z = 1891[Fig. 3(c)]. IBA was identified in fraction A, (molecular ion of methyl ester of IBA. M/Z = 217) [Fig. 6(b)]. No indole metabolites were identified by GC-MS in any of the fractions of the non-inoculated control.

DISCUSSION

In this work we found that a 72 h culture containing 2 x IO* cfu ml-’ of Azospiriflum produced 32dOng ml-’ of IAA when grown on tryptophanfree medium. A similar result was reported by Horemans and Vlassak (1985). When tryptophan was added to the medium the level of IAA was increased to 5Opg ml-’ (Jain and Patriquin, 1985). Bandurski and Schulze (1977) reported that the amount of conjugated and free IAA in vegetative tissue of maize was 328 and 24 ng g-’ fresh wt. respectively. line and Carr (1982) found 26 ng g-’ fresh wt of free IAA in 3-day-old etiolated maize shoots. The higher levels of endogenous IAA in the inoculated roots observed in our work (between 150-750 ng IAA g-’ fresh wt of conjugated IAA and I%--300 ng IAA g-’ fresh wt of free IAA) are probably a result of methods of cultivation, maize variety, growth conditions and duration of growth and sampling. Axupirillrun at IO’ cfu plant-’ significantly increased the root surface area from the second week on. It is clear that Axupirillum affect the amount of conjugated IAA in the inoculated roots of maize seedlings. Two weeks from the day of sowing there was a simultaneous decrease in the amount of conjugated IAA and a sharp, significant increase in the free IAA concentration compared to non-inoculated control. Moreover, after feeding of IAA to inoculated and non-inoculated seedlings, IAA and IBA were identified only in the roots of the inoculated ones. We can not exclude the possibility however that auxins were present in non-inoculated root extracts in amounts too low to be detected. IBA is a compound which is widely used in agriculture as a commercial promoter of root initiation in cuttings (Nickell. 1982) and is considered as a synthetic hormone. Recently IBA was identified by Schneider er al. (1985) as a endogenous constituent of pea tissues, and E. Epstein (unpublished) identified it in maize seeds and shoots. The presence of IBA in the inoculated

roots is clearly

demonstrated

in this work,

and may suggest that IBA alone, or jointly with IAA is involved in maize root proliferation of inoculated plants. When IAA is supplied to non-inoculated plant roots it is usually conjugated rapidly (Bandurski. 1980). The presence of free IAA in the inoculated roots which were fed with IAA for 24 h and its absence from the non-inoculated control may be attributed to hydrolysis of the conjugated IAA by the bacteria to release free IAA, or to blocking of conjugation of the exogenous IAA by the bacteria. It is still possible that the bacteria metabolize the plant tryptophan to IAA and excrete it into the plant as was reported with Pseudomonas syringae pv. sapastanoi (Morris, 1986).

Other rhizosphere bacteria have been reported to produce IAA (Morris, 1986; Prikryl et al., 1985) and to increase the amounts of IAA in plants. For example, Agrobacterium tumefaciens which causes crown gall, affects the amount of IAA directly via insertion of DNA-encoded phytohormone biosynthesis genes (Morris, 1986). In order to find out whether A:ospiriffum affects the plant by IAA excretion it would be necessary to isolate mutants which do not synthesize IAA and to study their effect on maize growth and function. The higher amount of free IAA and IBA which were found and identified in inoculated roots may be associated with the yield increase as described by Okon (1985). Acknowledgemenf-We would like to thank Dr James Anderson from The Plant Hormone Laboratory, USDA.ARS. Beltsville, Md, U.S.A. for the critical review of the manuscript.

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