Journal of Applied Phycology

, Volume 25, Issue 2, pp 379–386

Endogenous auxins in plant growth-promoting Cyanobacteria—Anabaena vaginicola and Nostoc calcicola

Authors

  • Mehri Seyed Hashtroudi
    • Department of Phytochemistry, Medicinal Plants and Drugs Research InstituteShahid Beheshti University
    • Department of Marine Living ScienceIranian National Institute for Oceanography
    • Department of Phytochemistry, Medicinal Plants and Drugs Research InstituteShahid Beheshti University
  • Hossein Riahi
    • Faculty of BiosciencesShahid Beheshti University
  • Zeinab Shariatmadari
    • Faculty of BiosciencesShahid Beheshti University
  • Maryam Khanjir
    • Faculty of BiosciencesShahid Beheshti University
Article

DOI: 10.1007/s10811-012-9872-7

Cite this article as:
Hashtroudi, M.S., Ghassempour, A., Riahi, H. et al. J Appl Phycol (2013) 25: 379. doi:10.1007/s10811-012-9872-7

Abstract

Three isolates of heterocystous cyanobacteria, belonging to the genera Anabaena and Nostoc, gathered from Iranian terrestrial and aquatic ecosystems exhibited considerable growth promotion effect on several vegetables and herbaceous plants. To study the ability of these three isolates to produce auxins, three endogenous auxins, including indole-3-acetic acid (IAA), and two of its main homologues, indole-3-propionic acid and indole-3-butyric acid, were extracted and analyzed with high-performance liquid chromatography equipped with diode array detector and fluorescence detector, and the results were further confirmed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the negative-ion mode. The dominant auxin observed in all isolates was indole-3-butyric acid (IBA) in the range of 140.10–2146.96 ng g−1 fresh weight (FW), and only small amounts of IAA (2.19–9.93 ng g−1 FW) were detected. The predominance of IBA in these strains is reported for the first time which is different from the previously reported auxin profiles in microalgae and algae with the predominance of IAA.

Keywords

Anabaena vaginicolaNostoc calcicolaIndole-3-butyric acid (IBA)Indole-3-acetic acid (IAA)Indole-3-propionic acid (IPA)HPLCLC–MS/MS

Introduction

Cyanobacteria or blue-green algae are the largest group of photosynthetic prokaryotes that exist in large diversity and distribution in the world (Stanier and Cohen-Bazire 1977). They are a rich source of potentially bioactive compounds like carotenoids, fatty acids, proteins, polysaccharides, vitamins and phenol compounds which exhibit antioxidant, anti-microbial, anti-inflammatory, hepatoprotective, immunomodulation and anti-cancer activity (Tan 2007; Ravi et al. 2010; Singh et al. 2011). The N2-fixing forms also improve the fertility of natural and cultivated ecosystems (Pardo et al. 2009; Maqubela et al. 2010). Increasing food demand has largely amplified the utilization of chemical fertilizers to achieve significant rise in crop yield. However, careless and overuse of fertilizers can lead to the pollution of both soil and water. Cyanobacteria offer an economically attractive and environmentally friendly alternative to chemical fertilizers which increase the soil productivity both directly and indirectly (Vaishampayan et al. 2001; Mishra and Pabbi 2004). Nostocacean cyanobacteria are broadly characterized by their unbranched filaments and the heterocysts which are the sites of nitrogen fixation (Komárek 2010). They are naturally found in most paddy soils and improve the fertility of soil at no cost (Bocchi and Malgioglio 2010). The influence of adding cyanobacteria to the soil to increase the rice yield has been known for many decades (Watanabe et al. 1951; Kulasooriaya et al. 1981; Roger 1982). Much attention has been paid to the study of nitrogen fixation of cyanobacteria in paddy fields since rice is the main food for the majority of people in the world. In a recent taxonomic study, soil samples were collected from 18 paddy fields of Iran and 33 isolates of heterocystous cyanobacteria were identified (Shariatmadari et al. 2011a). Plant growth promotion effect of two strains of these cyanobacteria, Anabaena vaginicola and Nostoc calcicola, was investigated on some vegetables and herbaceous plants including Solanum lycopersicum, Cucurbita maxima, Cucumis sativus (Shariatmadari et al. 2011b), and also Mentha spicata and Satureia hortensis (unpublished results) by treating them with algal extracts. The results revealed that in all experiments, there was a significant positive difference in most measurement factors. A similar study previously reported on rice (Saadatnia and Riahi 2009) and maize (Jäger et al. 2010).

The studies show that the nitrogen fixation capability is not the only factor contributing to the growth-promoting effect, and the existence of several other bioactive chemicals like plant growth regulators also play important role in this issue. Phytohormones such as auxins, cytokinins, abscisic acid, gibberellins, and ethylene are organic substances occurring in trace amounts in plants. They have many essential functions in regulating plant growth, metabolism, and development (Davies 2004). Auxin is a crucial phytohormone for the precise control of growth and development of plants (Taiz and Zeiger 1998). There are many indole conjugates with indole-3-acetic acid (IAA) being considered the most biologically active (Ludwig-Müller 2011). The aim of the present work was to identify and quantify the endogenous auxins in two strains of cyanobacteria, Anabaena vaginicola Fritsch et Rich and Nostoc calcicola Brébisson ex Bornet & Flahault in three Iranian isolates.

Material and method

The phytohormone standards, IAA and indole-3-butyric acid (IBA) were obtained from Duchefa Biochemie (Haarlem, Netherland), and indole-3-propionic acid (IPA) was purchased from Merck Chemicals (Darmstadt, Germany). All the solvents (HPLC gradient grade) were purchased from Caledon Lab (Ontario, Canada). Ultrapure water (from a Direct Q UV-3 Millipore system) was used in all experiments. The standard solutions were stored at 0–4 °C.

The stock solutions were prepared by dissolving 1 mg of each auxin in 10 mL methanol, separately and also as a mixture, and subsequent dilutions were made with methanol/water (80:20 v/v).

Extraction procedure

Two isolates of A. vaginicola from paddy soil of Gilan province and Miankaleh lagoon in Golestan province, and one isolate of N. calcicola from paddy soil of Mazandaran province of Iran were used for this study. Identification of these isolates was performed by morphometric and molecular methods (16S rRNA gene sequencing) (Shariatmadari et al. 2011a). Both the water and soil isolates were cultured in the same controlled conditions (environmental factors and culture medium). The algal stock cultures were maintained in solid BG-110 (nitrate-free) medium at 25 ± 2 °C under fluorescent illumination of 4,000 µmol photons m−2 s−1. Cultures were incubated in a growth chamber (12:12 h light/dark cycle). According to preliminary findings, harvesting was performed after 4 weeks. Due to the relatively similar growth rates of three isolates, cultures were harvested at the same time in their late logarithmic growth phase. The freeze-dried algal samples (25 mg dry weight) were extracted with 1 mL methanol/water (80:20 v/v) in a SONOREX DIGITEC DT 103 H ultrasonic bath (Bandelin, Germany) with an ultrasonic frequency of 35 kHz and an effective ultrasound power of 140 W for 30 min at 15 °C. The extracts were then centrifuged at 6,728 × g for 10 min at 15 °C. The supernatant was filtered through a 0.45-μm PTFE syringe filter and concentrated to 500–1,000 μL using a nitrogen evaporator.

HPLC

Chromatographic separation was performed on an Agilent 1200 series high-performance liquid chromatography (HPLC) system including a quaternary pump and a degasser equipped with a G1315D diode array detector and a G1321A fluorescence detector. The accompanying Agilent LC Chemstation was used for instrument control, data acquisition, and processing. Preliminary experiments showed that the best results were obtained with the Eurosphere reverse phase column (100-5 C18 column, 250 × 4.6 mm; Knauer, Germany) and this was used for the analysis of subsequent extracts. The column was eluted with a linear gradient (0–5 min, 60 % A, 5–20 min, 100 % A) at a flow rate of 1 mL min−1 methanol (A) and water and 0.3 % acetic acid (B); the column temperature was maintained at 25 °C. Considering the UV maxima of three auxins, UV detection was at 225 nm and excitation and emission wavelengths in the fluorescence detector were 280 and 360 nm, respectively.

Both the extracts and standards were injected (injection volume: 20 μL) into the reverse phase column, and identifications were carried out using comparison of retention times, UV spectra of the extracts with standard mixture, and also analysis of the samples spiked with auxin standards. Each experiment was repeated at least three times and run in triplicate. Recoveries were calculated by adding a known amount of standards to the cyanobacterial samples and extracting the auxins with the same method as described above.

LC–MS/MS conditions

LC/MSD (Model 1200 Series, Agilent Technologies coupled to a quadrupole ion trap Finnigan LCQ spectrometer) with electrospray ionization (ESI) and atmospheric pressure chemical ionization interfaces were used in scan mode. The better results were obtained with ESI. ESI–MS analysis was performed in both positive and negative ion modes, and the ion trap was scanned at m/z 50–500 in full scan and multiple reaction monitoring (MRM) modes. The optimum mass conditions were as follows: capillary voltage 4.5 kV; nebulizer pressure 40 psi; drying gas flow 8 L min−1; temperature 220 °C; m/z 50–500. The whole gradient program was the same as HPLC except for the flow rate which was 0.7 mL min−1 .The MRM mode was used to monitor the transitions from the precursor ions to the most abundant product ions. Table 1 shows the structural information and characteristic ions of three auxins. Both standard mixture and the three samples were separately analyzed by LC–MS/MS in the above-mentioned conditions.
Table 1

Structural information and characteristic ions of the three auxins

Auxin

Molecular weight

Precursor ions (m/z)

Fragment ions (m/z)

IAA

175

174

130

IPA

189

188

144, 116

IBA

203

202

184, 158, 116

Results

The microscopic images of the two strains used in this study including N. calcicola and A. vaginicola show their filaments and heterocysts which are the sites of nitrogen fixation (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9872-7/MediaObjects/10811_2012_9872_Fig1_HTML.gif
Fig. 1

aN. calcicola ISC89; bA. vaginicola ISC90, the heterocysts are marked with arrows

Figure 2 shows the HPLC chromatograms of the simultaneous analysis of a standard mixture of three auxins in a single run with diode array detector (DAD) and fluorescence detector (FLD).
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9872-7/MediaObjects/10811_2012_9872_Fig2_HTML.gif
Fig. 2

HPLC chromatograms of a 250 ng mL−1 standard of three auxins, with diode array detector (dashed line) and fluorescence detector (solid line). HPLC conditions: a linear gradient (0–5 min, 60 % A, 5–20 min, 100 % A) at a flow rate of 1mL min−1 methanol (A) and water and 0.3% acetic acid (B), C18 reversed phase column: 250 mm × 4.6 mm, 5 μm. UV detection wavelength at 225 nm, fluorescence excitation and emission wavelengths at 280 and 360 nm, respectively, column temperature of 25 °C

The retention times of each auxin were 5.287, 6.990, and 9.127 min for IAA, IPA, and IBA, respectively. The reproducibility of the retention times of the three auxins in this condition was investigated with doing repeated injections (n = 5) of the standard mixture at the concentration of 100 ng mL−1. The relative standard deviations (RSDs) of the retention times for all analytes were in the range of 0.45–0.85 %. The calibration curves were also prepared with five concentrations (10, 25, 50,100, and 500 ng mL−1) of the standard mixture. A linear correlation was found between concentration and the peak area for the three auxins in the range of calibration concentrations; typically R2 values were in the range of 0.992–0.998. The recoveries of three auxins were 84–86 %. The equation of calibration curves, R2, RSDs, and recoveries are summarized in Table 2.
Table 2

Equation of calibration curves, R2 values, precisions, and recoveries of the three auxins

Auxin

Equation of calibration curve

R2

RSD (%)

Recovery (%)

IAA

y = 0.1225x + 6.6179

0.992

0.58

84

IPA

y = 0.0729x + 2.6363

0.998

0.85

86

IBA

y = 0.0697x + 4.0452

0.996

0.45

85

The HPLC chromatograms of the three microalgal samples under the optimized HPLC conditions are shown in Fig. 3. The peaks were identified by comparing the retention times of authentic standards, the UV spectra, and also spiking the individual standards to the microalgae extracts. The observed peaks at 9.130, 9.147, and 9.288 min were related to IBA, when compared to the peak of IBA in the chromatogram of standard mixture at 9.127 min. Table 3 shows the estimated concentrations of the three auxins in the microalgal samples after considering the recoveries, with the highest amount in A. vaginicola from water 2,146.96 ng g−1 fresh weight (FW), 140.1 and 294.29 ng g−1 FW in A. vaginicola and N. calcicola (soil isolates). Only small amounts of IAA, 9.93, 2.19, and 4.57 ng g−1 FW, were observed in the samples.
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9872-7/MediaObjects/10811_2012_9872_Fig3_HTML.gif
Fig. 3

HPLC chromatograms of the ultrasonicated samples for 30 min (a) N. calcicola, (b) A. vaginicola (water isolate) and (c) A. vaginicola (soil isolate). The HPLC running condition is the same as Fig. 4

Table 3

Estimated concentrations of the three auxins in the microalgal samples

 

Estimated concentration (ng g−1) FW

IAA

IPA

IBA

Anabaena vaginicola (water)

9.93

ND

2146.96

Anabaena vaginicola (soil)

2.19

ND

140.1

Nostoc calcicola (soil)

4.57

ND

294.29

ND not detected

To ensure and further confirm the identities of the phytohormones in the real samples with more interference from other analytes, LC–MS/MS was performed. The mass spectra were obtained in full scan and MRM modes. The scan range was from m/z 50 to 500. The negative ions of IAA, IPA, and IBA showed major and strong mass peaks at m/z 174, 188, and 202, respectively. Figure 4 shows full mass and MS/MS spectra of the three auxins. The fragmentation pathways have also been shown in the spectra.
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9872-7/MediaObjects/10811_2012_9872_Fig4_HTML.gif
Fig. 4

The MS spectra of 10 μg mL−1 auxin standards from a single LC-MS run in the negative ion mode. Left: characteristic full MS spectra of IAA, IPA and IBA. The optimum Mass conditions: capillary voltage 4.5 kV; nebulizer pressure 40 psi; drying gas flow 8 L/min; temperature 220 °C; m/z 50–500. The whole LC gradient program was the same as HPLC except for the flow rate which was 0.7 mL min−1. Right: ES-MS/MS spectra of the precursor ions of m/z 174, 188 and 202

Application of the method to the three samples of cyanobacteria also confirmed the dominant presence of IBA in the samples with only small amounts of IAA detected. No IPA was detected in any of the samples. Figure 5 shows the MS spectrum of the phytohormones in the A. vaginicola (water).
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9872-7/MediaObjects/10811_2012_9872_Fig5_HTML.gif
Fig. 5

The MS spectra of A. vaginicola (water isolate) in a single LC-MS run in the (a) full scan mode (b) MRM mode. The optimum Mass conditions were the same as described in Fig. 4

Discussion

The observed predominance of Anabaena and Nostoc in the cyanobacteria from paddy soils of seven cultivating provinces in Iran made them good candidates for conducting experiments to investigate their growth-promoting effect (Shariatmadari et al. 2011a). The most widely studied auxin is IAA. IAA was determined in the culture medium of two axenic green microalgae—Chlorella pyrenoidosa and Scenedesmus armatus (Mazur et al. 2001). Production of indole-3-acetic acid was also reported in cyanobacteria using ELISA and further verification with GC–MS (Sergeeva et al. 2002). The assay of IAA in 16 Chinese marine algae (seaweeds) using wheat coleoptile bioanalysis and fluorescence spectrophotometry showed the concentrations 5.3–110.2 and 1.1–46.9 ng g−1 FW, respectively (Lijun 2006). It was also shown that addition of tryptophan as the IAA precursor to the culture media could increase the accumulation of IAA in the cyanobacteria. Gutierrez reported IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment for the first time (Gutierrez et al. 2009). Of course, IAA production was not detectable in the culture media lacking tryptophan. The reported work on the production of IAA by the cyanobacterium Arthrospira platensis was also completely tryptophan-dependent (Ahmed et al. 2010). In a recent work, IAA production in two strains of cyanobacteria was calorimetrically identified, and its production was stimulated by adding supplementary tryptophan (Mazhar and Hasnain 2011). But, in our study, no exogenous tryptophan was added to the culture media.

One of the major concerns involving measurement of auxins is the trace amounts of these hormones in the plants. So, the first step for a successful determination of them was selecting the most proper analysis method. In the present work, a HPLC equipped with DAD and FLD was selected for the analysis of auxins which did not require the methylation step in the gas chromatography–mass spectrometry (GC–MS) measurement. IAA and two of its main homologues, IPA and IBA, were separated with a good resolution in a short time of 9 min. Figure 2 shows obviously that the FLD is more sensitive than DAD in the analysis of auxins and therefore, it was used for the quantification in the real samples. To make certain of a correct identification, a known amount of auxin standards was added to the extracts, and the spiked samples were injected to HPLC. The intensities of supposed peaks of IBA and IAA were increased, which approved the identity of the IAA and IBA in the extracts. A comparison of all the results leads us to conclude the predominance of IBA in the investigated cyanobacteria in relatively high concentrations. The highest concentration was observed in the A. vaginicola obtained from Miankaleh. N. calcicola and A. vaginicola, the isolates gathered from paddy soils, have lower concentrations of auxins but still with predominance of IBA. IAA was detectable only in small quantities in all three isolates, and no IPA was detected in any of the samples.

To confirm the obtained results, LC–MS/MS with ESI interface was also performed in both positive and negative ion modes. The negative ion mode showed better selectivity and higher intensities for these auxins. As it could be seen in Fig. 4, in the negative ion mode, IAA, IPA, and IBA which are weak acidic organic compounds are dissociated and show major and strong mass peaks at m/z 174, 188, and 202, respectively, which are referred to the precursor ions [M–H]. The most probable fragmentation pathways of precursor ions of three auxin standards are shown in Fig. 4. In IAA, IPA, and IBA, a carboxyl group is firstly dissociated to give major fragments at m/z 130, 144, and 158, respectively. The m/z 116 could be attributed to the loss of the side chain hydrocarbon on the third carbon of the indole ring in IPA and IBA which produces the stable indole ring fragment with a negative charge. This fragment is not present in IAA. IBA can also loose a H2O moiety to produce a peak at m/z 184.

From the results of the LC–MS/MS investigation, the typical mass spectra related to A. vaginicola (water specimen) in Fig. 5a showed the precursor ion at m/z 202 relevant to [M–H] and the major fragment at m/z 184, 158, and 116, which are identical with authentic IBA spectra in full scan and MRM mode.

Thus, our results confirmed the predominance of IBA in the three Iranian isolates of cyanobacteria which is somewhat different with the previously reported results in which the predominant auxin was IAA, especially when tryptophan was used as a precursor. In our experiments, monoalgal culture was performed in a nitrate-free medium. It is also important to mention that the algal cultures were harvested at their late logarithmic growth phase, because the cyanobacterial samples showed the maximum accumulation of auxins after this time period, meanwhile enough algal biomass was obtained for studying of plant growth-promoting effect.

Until the late 1980s, IBA was thought to be strictly synthetic, but later, it was reported that this auxin was isolated from leaves and seeds of maize and other species (Epstein et al. 1989). This has been confirmed with comprehensive studies on the occurrence, biosynthesis, metabolism, and transport of IBA in the plants, especially in Zea mays L. and Arabidopsis thaliana (Ludwig-Müller and Epstein 1991, 1992, 1994; Ludwig-Müller 2000). IBA was found to be considerably more effective than IAA (Blazich 1988) at promoting adventitious root formation. The reason could be because of less susceptibility of IBA to the degradative enzymes than IAA, the slow conversion of IBA to IAA providing a steady supply of free IAA for the plant (Strader et al. 2010), or the higher stability of IBA over IAA both in solution and in plant tissues (Nordström et al. 1991). There is still little known about the actual role of auxins in algal lineage (Lau et al. 2009; Spaepen et al. 2007). However, it seems that similar to higher plants, IBA may be converted to IAA to maintain the auxin level, and it has a possible role as an auxin storage form (Bertoni 2011). So, it appears that all of our Iranian isolates belonging to the genera Anabaena and Nostoc, are capable of producing auxins especially IBA in relatively high concentrations which beside the trace amounts of IAA could contribute to the observed growth promotion effect on our studied plants.

In conclusion, we report here the determination and quantification of three auxins (IAA, IPA, and IBA) in three Iranian isolates belonging to heterocystous cyanobacteria, Anabaena and Nostoc, for the first time. All isolates showed considerable growth-promoting effect on several vegetables and herbaceous plants. Simultaneous analysis of auxins was performed using a relatively simple HPLC method with high precision within a short time, and no preliminary purification was required. The results showed the predominance of IBA in all three isolates which is different from previously reported concentrations of auxins for algae and cyanobacteria.

Acknowledgments

The financial support by Shahid Beheshti University Research Council and Iranian National Institute for Oceanography (INIO) is highly acknowledged. Special thanks to Dr J. H. Gross and Prof O. Trapp (University of Heidelberg, Institute of organic chemistry) whose kind assistances have been invaluable in this project.

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