Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

Abstract

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.

Full text

ORIGINAL ARTICLE
Elena Sergeeva Æ Anton Liaimer Æ Birgitta Bergman
Evidence for production of the phytohormone indole-3-acetic
acid by cyanobacteria
Received: 8 October 2001 / Accepted: 11 January 2002 / Published online: 22 March 2002
 Springer-Verlag 2002
Abstract The ability of cyanobacteria to produce the
phytohormone indole-3-acetic acid (IAA) was demon-
strated. A colorimetric (Salkowski) screening of 34 free-
living and symbiotically competent cyanobacteria, that
represent all morphotypes from the unicellular to the
highly differentiated, showed that auxin-like compounds
were released by about 38% of the free-living as com-
pared to 83% of the symbiotic isolates. The endogenous
accumulation and release of IAA were confirmed im-
munologically (ELISA) using an anti-IAA antibody on
10 of the Salkowski-positive strains, and the chemical
authenticity of IAA was further verified by chemical
characterization using gas chromatography-mass spect-
rometry in Nostoc PCC 9229 (isolated from the angio-
sperm Gunnera) and in Nostoc 268 (free-living).
Addition of the putative IAA precursor tryptophan en-
hanced IAA accumulation in cell extracts and superna-
tants. As the genome of the symbiotically competent
Nostoc PCC 73102 contains homologues of key enzymes
of the indole-3-pyruvic acid pathway, a transaminase
and indolepyruvate decarboxylase (IpdC), the putative
ipdC gene from this cyanobacterium was cloned and
used in Southern blot analysis. Out of 11 cyanobacterial
strains responding positively in the Salkowski/ELISA
test, ipdC homologues were found in 4. A constitutive
and possibly tryptophan-dependent production of IAA
via the indole-3-pyruvic acid pathway is therefore sug-
gested. The possible role of IAA in cyanobacteria in
general and in their interactions with plants is discussed.
Keywords Cyanobacterium Æ Indole-3-acetic acid Æ
Indolepyruvate decarboxylase Æ Plant
symbiosis Æ Tryptophan
Abbreviations Chl a: chlorophyll a Æ ELISA: enzyme-
linked immunosorbent assay Æ IAA: indole-3-acetic
acid Æ IpdC: indolepyruvate decarboxylase Æ TPP:
thiamine pyrophosphate Æ Trp: tryptophan
Introduction
Cyanobacteria are prokaryotic photoautotrophs of
evolutionary significance (Schopf 2000). They rely on an
oxygenic photosynthesis involving chlorophyll, photo-
system I/II electron transport and CO
2
fixation (Fay
1992). Cyanobacteria range in their morphology from
unicellular to differentiated, and branched types and are
widespread in nature.
Cyanobacteria also constitute a direct evolutionary
link between pro- and eukaryotes as cyanobacteria were
progenitors of chloroplasts (Douglas 1994). The en-
gulfment of a cyanobacterium by a primitive pigment-
less ancestral eukaryote led to the genesis of plants with
photosynthetic chloroplasts. Some cyanobacteria, par-
ticularly within the genus Nostoc, still enter into intimate
relationships with some eukaryotic organisms repre-
senting all plant divisions (Rai et al. 2000). Hosts are
found within liver- and hornworts, a water-fern (Azolla),
cycads and one angiosperm, as well as some fungi
(primarily lichenized fungi) and protists. The primary
benefit for the plants may be ascribed to the nitrogen-
fixation capacity of the cyanobiont, as most of the fixed
nitrogen is transferred to the host. However, the in-
volvement of other cyanobacterial molecules promoting
growth and development of host plants is also a possi-
bility in cyanobacterial–plant interactions, although
none has been identified.
Phytohormones play an important role as signals and
regulators of growth and development in plants. Auxins,
among them in particular indole-3-acetic acid (IAA), are
the most studied plant growth regulators, and this in-
cludes physiological, biochemical and genetic aspects.
The ability to produce the phytohormones is often
considered as a trait of the plant kingdom. However,
Planta (2002) 215: 229–238
DOI 10.1007/s00425-002-0749-x
E. Sergeeva Æ A. Liaimer Æ B. Bergman (&)
Department of Botany,
Stockholm University, 10691 Stockholm, Sweden
E-mail: bergmanb@botan.su.se
Fax: +46-8-165525

production of phytohormones is also widespread among
soil and plant-associated prokaryotes (Costacurta and
Vanderleyden 1995). Phytohormones produced by
plant-associated bacteria are implicated as key deter-
minants in stimulation of plant growth, in plant patho-
genesis and in associative or plant–microbe symbiotic
interactions (Christiansen-Weniger 1998). As some
plant-interacting bacteria release high levels of IAA,
they may locally change the endogenous phytohormone
balance of the host and trigger responses, the nature of
which depends on the phytohormone concentration
generated, type of host and its sensitivity to hormones
(Glick et al. 1999). Known plant growth-promoting
bacteria include species within genera such as Herbas-
pirillum, Rhizobium and Azospirillum, interacting with
plant roots, while for instance Pseudomonas, Erwinia
and Agrobacterium elicit galls or tumours on plants.
Possibly, phytohormone release provides plant-inter-
acting bacteria with a tool by which to promote plant
cell division, growth and nutrient release and thereby
their own growth. Genetic mechanisms underlying IAA
biosynthesis and its regulation have been studied in
Pseudomonas, Agrobacterium, Rhizobium, Bradyrhizobi-
um and Azospirillum, and multiple pathways for IAA
synthesis have been identified, including both trypto-
phan-dependent and independent pathways (Glick et al.
1999).
Up to now, there has been no unequivocal identifi-
cation of phytohormone production in cyanobacteria.
The aim of the present study was to examine the po-
tential in cyanobacteria to produce the phytohormone
IAA. This was done by screening for endogenous IAA
levels and IAA release in a range of free-living cyano-
bacteria, belonging to various morphotypes, and also in
symbiotically competent cyanobacteria. We demonstrate
that cyanobacteria, and in particular symbiotic isolates,
are capable of both accumulating and releasing IAA,
and suggest that IAA accumulation is stimulated by
exogenous tryptophan and may proceed via the indole-
3-pyruvic acid pathway.
Materials and methods
Organisms and growth conditions
The 34 species of cyanobacteria examined were obtained from the
sources given in Table 1 and all are maintained in our culture
collection (Department of Botany at Stockholm University). The
cyanobacteria were re-inoculated every second week and were
grown axenically in either nitrogen-free BG-11
0
medium (for ni-
trogen-fixers) or, for cyanobacteria incapable of fixing nitrogen, in
BG-11 medium with 1.5 g l
–1
of NaNO
3
added (Stanier et al. 1971).
The cultivation temperature was 28 C, and the specific conditions
of light and shaking were as given by Johansson and Bergman
(1994).
Detection of auxin-like substances with the Salkowski reagent
Cultures grown for 18 days were centrifuged at 5,000 g for 20 min
at 4 C and then supernatant was purified using a C
18
Sep-Pack
cartridge (Waters, Eschborn, Germany). The culture supernatant
was used for determinations of released, Salkowski-positive indolic
compounds, likely to represent the auxin (Glickmann and Dessaux
1995). The Salkowski reagent, modified according to Pilet and
Chollet (1970), was added to the supernatant in a ratio of 1:1 (v/v).
Concentrations of auxin-like substances were estimated by absor-
bance at 530 nm after 30 min in the dark at room temperature
against a control containing 1 ml of culture medium and 1 ml of
Salkowski reagent.
Extraction and purification
Supernatants and cell extracts of the 10 selected cyanobacteria were
used for detection and estimation of concentrations of IAA in the
cyanobacteria. Cells were harvested by centrifugation at 5,000 g for
20 min at 4 C. The culture supernatant was adjusted to pH 2.8
with 1.0 M HCl and extracted three times with ethyl acetate (1:3, v/
v). Extracts were then evaporated under vacuum at 37 C (Buchi,
Flawil, Switzerland). The aqueous fraction was adjusted to pH 7.0
with 1.0 N NaOH and extracted three times with water-saturated
n-butanol followed by drying in vacuum. The extracts obtained
were filtered through membrane filters (pore size 0.45 lm). Har-
vested cyanobacterial cells were homogenised in liquid nitrogen
using a cold mortar and pestle. The resulting powder was extracted
overnight in 80% methanol containing 10 mg l
–1
butylated hy-
droxytoluene as antioxidant at 4 C. The methanolic fractions were
centrifuged at 4,000 g for 10 min at 4 C, followed by filtering
through glass microfibre filters (pore size 1.2 lm; GF/C, What-
man). The filtrate was then passed through a C
18
Bond Elut solid-
phase extraction column (Varian, Palo Alto, Calif., USA) and
eluted with 80% methanol. Eluates were partitioned with ethyl
acetate and water-saturated butanol as described above. The or-
ganic phases were reduced to dryness with nitrogen and the resi-
dues were re-dissolved in methanol, diluted with 1% acetic acid and
applied to C
18
columns as described above. All extracts were stored
at –18 C and assayed within 48 h.
Enzyme-linked immunosorbent assay (ELISA)
Samples extracted from cells and culture supernatants of cyano-
bacteria were methylated with approximately 1.5 ml of diluted
diazomethane (Cohen 1984) for 5 min at 0 C and dried under
nitrogen. An ELISA test (Phytodetek IAA; Agdia, Elkhart, Ind.,
USA) was used according to the manufacturer’s instructions to
identify IAA. The anti-IAA monoclonal antibody contained in this
kit is documented to show a high specificity for IAA in the
methylated form. The production and characterisation of the high-
affinity monoclonal antibody was as described by Mertens et al.
(1985).
GC-MS analysis
[
13
C
6
]Indole-3-acetic acid (1 ng; 99% enrichment; Cambridge
Isotope Laboratories, Woburn, Mass., USA) as an internal stan-
dard was added before the procedure for extraction and purifi-
cation of IAA from the cyanobacteria mentioned above. Samples
were methylated with diazomethane and dissolved in heptane.
GC-MS analyses were performed according to Edlund et al.
(1995). All samples were processed by the JEOL MS-MP7000 D
data system.
Chlorophyll measurement
The Nostoc cells were collected by centrifugation, the pellets were
ground in liquid nitrogen and the resulting homogenate was dis-
solved in 80% methanol. After 2 h incubation in darkness, cell
debris was removed by centrifugation and the absorbance of the
supernatant was measured against 80% methanol as a blank.
Chlorophyll content (lgml
–1
) was determined from the optical
230

density at 665 nm (OD
665nm
), calculated as OD
665nm
·13.9
(Tandeau de Marsac and Houmard 1988).
Similarity search
Preliminary sequence data of Nostoc punctiforme ATCC 29133 and
Synechococcus WH8102 were obtained from the DOE Joint Ge-
nome Institute (JGI) at http://spider.jgi-psf.org/JGI_microbial/
html ,and Nostoc sp. PCC 7120, Synechocystis sp. PCC 6803 from
the Kazusa DNA Research Institute at http://www.kazusa.or.jp/
cyano/cyano.html. Similarity searches were performed using
BLAST (Altschul et al. 1997) provided at the sites listed above or
included in the Biology Workbench 3.2 package from the San
Diego Supercomputer Center at http://workbench.sdsc.edu. Mul-
tiple sequence alignments were done using CLUSTAL W
(Thompson et al. 1994). The full-length deduced amino
acid sequences of indole-3-pyruvate decarboxylases (IpdCs) from
Azospirillum brazilense, Enterobacter cloacae, Erwinia herbicola and
Pseudomonas putida, tryptophan monooxygenases (IaaMs) and
indole-3-acetamide hydrolases (IaaHs) from Pseudomonas syringae
and Agrobacterium vitis were obtained from the GenBank at the
National Center for Biotechnological Information, NCBI (Gen-
Bank accession numbers P51852, BAA14242, AAB06571,
AAG00523, M11035, M35690, AF061780, AF039169).
DNA analysis
A 1805-bp fragment of the putative ipdC gene from Nostoc sp.
PCC 73102 was PCR-amplified with DynaZyme (Finnzymes,
Espoo, Finland). The primers used were: IPDC-F 5¢-AACGG-
CAAAAGTCACATTCC-3¢ and IPDC-R 5¢-TGAGTTGAC-
GAGGCAGTACG-3¢. The PCR product was ligated into the
Table 1 Screening of release of auxin-like indolic substances from
free-living and symbiotic cyanobacteria using the Salkowski re-
agent. The results are from three independent experiments (after
18 days of cultivation). Symbols represents colour intensity:
+++, strong; ++, average; +, weak; –, no reaction. The true
identity of the isolate denoted Nostoc/Anabaena azollae isolated
from Azolla leaf cavities has been questioned (see Rai 1996)
a
U Unicellular cyanobacteria, NH filamentous non-heterocystous,
FH filamentous heterocystous, N
+
capable of fixing atmospheric
nitrogen, Ex/In extra- or intracellular location of the cyanobacte-
rium in the host
b
Collection Nationale de Cultures de Microoganismes, Institut
Pasteur, Paris (France)
c
Department of Microbiology, University of Helsinki, Helsinki
(Finland)
d
Department of Botany, Stockholm University, Stockholm (Swe-
den)
e
Division of Microbiology, University of Leeds, Leeds (UK)
f
Department of Microbiology, University of California, Davis
(USA)
Cyanobacterial genera Cyanobacterial
species/strain
Morphotype/host plant
a
Auxin-like
Salkowski reaction
Source
Free-living:
Synechocystis PCC 6803 U – CNCM
b
Anacystis PCC 6301 U – CNCM
b
Gloeothece PCC 6909 U/N
+
+ CNCM
b
Phormidium NH – B. Bergman
d
Plectonema PCC 73110 NH/N
+
++ CNCM
b
Symploca PCC 8002 NH/N
+
– CNCM
b
Calothrix PCC 7504 FH/N
+
+++ CNCM
b
Calothrix PCC 7103 FH/N
+
+++ CNCM
b
Scytonema PCC 7110 FH/N
+
– CNCM
b
Chlorogloeopsis PCC 6912 FH/N
+
– CNCM
b
Fischerella PCC 7521 FH/N
+
– CNCM
b
Nostoc 268 FH/N
+
+ T. Vaara
c
Nostoc/Anabaena PCC 7120 FH/N
+
– CNCM
b
Nostoc PCC 6720 FH/N
+
– CNCM
b
Nostoc PCC 6310 FH/N
+
+ CNCM
b
Nostoc PCC 7107 FH/N
+
– CNCM
b
Symbiotic/N
+
:
Nostoc Pc Lichen (Ex) +++ B. Bergman
d
Nostoc LBG1 Liverwort (Ex) + D. Adams
e
Nostoc PCC 9305 Liverwort (Ex) + CNCM
b
Nostoc 7801 Hornwort (Ex) + J.C. Meeks
f
Nostoc/Anabaena azollae Newton’s isolate Water-fern (Ex) +++ J.C. Meeks
f
Nostoc 8071 Cycad (Ex) + J.C. Meeks
f
Nostoc MacS Cycad (Ex) + CNCM
b
Nostoc PCC 7422 Cycad (Ex) – CNCM
b
Nostoc PCC 73102 Cycad (Ex) ++ CNCM
b
Nostoc 8005 Gunnera (In) – J.C. Meeks
f
Nostoc Chr 891 Gunnera (In) + C. Johansson
d
Nostoc Chr 892 Gunnera (In) ++ C. Johansson
d
Nostoc Chr 893 Gunnera (In) + C. Johansson
d
Nostoc Chr 894 Gunnera (In) +++ C. Johansson
d
Nostoc 7901 Gunnera (In) + J.C. Meeks
f
Nostoc 8001 Gunnera (In) ++ J.C. Meeks
f
Nostoc PCC 9229 Gunnera (In) ++ CNCM
b
Nostoc PCC 9231 Gunnera (In) – CNCM
b
231

pCR 2.1 vector and cloned in Escherichia coli TOP 10F¢ (Invitro-
gen, Groningen, The Netherlands). The insert obtained was used as
a probe in Southern analysis. Total DNA from 11 cyanobacterial
strains (Gloeothece sp. PCC 6909, Plectonema sp. PCC 73110,
Nostoc sp. PCC 6720, Nostoc sp. PCC 7120, Nostoc sp. 268, Nostoc
sp. Pc, Nostoc sp. LBG1, Nostoc sp. PCC 73102, Nostoc sp.
Chr 894, Nostoc sp. PCC 9229 and Nostoc/Anabaena azollae) was
isolated as in Cohen et al. (1994). For each strain, total DNA (8–
10 mg) was digested with EcoRI (Amersham Pharmacia, Uppsala,
Sweden) and separated on a 1% agarose gel with 1·TAE buffer
(Sambrook et al. 1989). The DNA was transferred onto Hybond
N+ membrane (Amersham Pharmacia). Southern hybridisation
was performed with the ECL direct nucleic acid labelling and de-
tection system (Amersham Pharmacia) according to the manufac-
turer’s instructions.
Results
Screening for the release of auxin-like indolic
compounds
As a first step, 34 free-living and symbiotically compe-
tent cyanobacterial strains were screened for their ability
to produce auxin-like substances (Table 1). Represen-
tatives from all major cyanobacterial morphotypes were
analysed: unicellular (U), filamentous non-heterocystous
(NH) and filamentous heterocystous (FH; heterocysts
are cells that fix atmospheric nitrogen). Moreover, the
selection embraced cyanobacterial strains with known
symbiotic competence, originally isolated from five host-
plant groups, from a lichenized fungus to the angio-
sperm, Gunnera.
The auxin-like substances were determined using the
Salkowski reagent. As this reagent represents a simple
and fast assay, and as it is known to recognise indolic
compounds such as IAA (Glickmann and Dessaux 1995),
it was used for the determination of IAA by Hartman
et al. (1983) and Zimmer et al. (1991). Twenty-one of the
34 cyanobacterial strains tested (approx. 62%) showed a
positive colour reaction indicative of a release of an IAA-
like substance into the culture supernatant (Table 1). Of
the 16 free-living strains, about 38% released IAA-like
substances. Positive representatives were found among
all three cyanobacterial morphotypes (U, NH, FH;
Table 1), and the nitrogen-fixing heterocystous genus
Calothrix (PCC 7103 and PCC 7504) appeared particu-
larly potent. No correlation between release of IAA-like
substances and morphological complexity was detected.
The most complex representatives (Scytonema, Chloro-
gloeopsis and Fischerella), belonging to group-V cyano-
bacteria sensu Rippka et al. (1979), showed no
Salkowski-positive response. Neither was there any cor-
relation between the Salkowski response and nitrogen-
fixation capacities (Table 1).
Of the 18 symbiotic Nostoc strains (all belonging to
group-IV cyanobacteria), 83% released IAA-like sub-
stances. The highest levels of release were seen in Nostoc
isolated from the lichenized fungus Peltigera, from the
angiosperm Gunnera and the putative isolate from the
water-fern Azolla, but all host divisions were represented
among Salkowski-positive strains. There was no corre-
lation between release of IAA-like substances and
whether the cyanobacterium forms an intra- or extra-
cellular (In/Ex) symbiosis (Table 1).
Immunological identification of IAA
To obtain a more accurate qualitative identification and
quantitative determinations of the IAA released as well
as of the cellular IAA contents, an immunological ap-
proach was used. Ten of the screened cyanobacteria
(Table 1) were selected for examination using a specific
monoclonal anti-IAA antibody using ELISA (Weiler
et al. 1981). This assay detects the IAA methyl ester
down to 10–20 fmol, and is claimed to be as sensitive as
GC-MS (Pengelly et al. 1981). As is obvious from
Table 2, an IAA-positive response was obtained from
the strains examined, although the quantities varied,
with the unicellular Gloeothece PCC 6909 accumulating
and releasing the least. On average, both the endogenous
and exogenous IAA levels were of the same order of
magnitude within the two groups (Table 2). In general,
Table 2 Endogenous and
exogenous levels of IAA
produced by cyanobacteria
grown in the presence and
absence of tryptophan (Trp) as
evidenced by an ELISA test.
The tests were performed after
2 weeks of cultivation. The
values represent the mean ±
SD from two independent ex-
periments with three replicates
each
Cyanobacterial strain
a
Endogenous IAA Exogenous IAA
(pmol mg
–1
Chl a) (pmol mg
–1
Chl a)
–Trp +Trp –Trp +Trp
Free-living:
Gleoethece 3.5±0.6 3.3±0.3 1.2±0.5 1.2±0.6
Plectonema 12.8±0.5 14.9±0.2 12.6±0.6 10.5±0.5
Nostoc 268 10.1±0.4 30.9±0.8 5.2±0.3 9.1±0.4
Nostoc 7120 15.3±0.8 21.1±0.5 9.9±0.6 12.6±0.4
Nostoc 6720 6.0±0.4 20.8±1.1 11.3±0.7 19.5±0.3
Symbiotic:
Nostoc Pc 16.2±0.5 26.8±0.7 23.5±0.9 25.7±1.2
Nostoc/Anabena 12.5±0.5 20.7±1.4 10.1±0.8 18.6±1.1
Nostoc 73102 24.2±1.1 42.2±1.5 19.9±0.7 34.0±1.2
Nostoc Chr 894 7.2±0.6 37.6±1.6 15.2±0.8 24.7±0.9
Nostoc 9229 14.9±0.6 28.5±0.7 8.2±0.3 23.1±0.6
a
For morphotype, strain identification and source of the cyanobacteria used, see Table 1
232

the symbiotic isolates showed higher endogenous IAA
levels and released more IAA than the free-living
counterparts.
Tryptophan (Trp) is considered as the main precursor
for the biosynthesis of IAA in plants and microorgan-
isms, but with several possible pathways and interme-
diates involved in generating the final product, IAA.
Adding 500 mg l
–1
Trp to the cyanobacterial cultures
stimulated the endogenous accumulation of IAA about
2-fold in both free-living and symbiotic strains, while
IAA release was less enhanced by Trp (Table 2). Nostoc
PCC 73102 accumulated about 42 pmol and released
34 pmol IAA per mg chlorophyll a (Chl a) after 2 weeks
in the presence of Trp, the highest quantities noted.
Accumulation and release of IAA
in two Nostoc strains
Two of the Nostoc strains were selected for comparative
analyses of their IAA production during a 3-week
growth cycle. The cyanobacteria were the free-living
Nostoc 268, originally isolated from the Baltic Sea and
not known to be involved in any symbiotic interactions
and incapable of infecting Gunnera (Johansson and
Bergman 1994), and the symbiotically competent Nos-
toc 9229. The latter readily infects Gunnera cells intra-
cellularly. Both Nostoc strains continuously
accumulated IAA endogenously and released IAA dur-
ing the 3-week growth period, and the IAA concentra-
tions in the free-living Nostoc 268 were in general lower
than in the symbiotic isolate (Fig. 1a, b, Table 2).
However, IAA was not detected after 24 h of growth,
neither was a release seen in the absence of Trp within
72 h. Maximum endogenous IAA levels for Nostoc 9229
were about 20 pmol IAA per mg Chl a and that of IAA
release about 15 pmol per mg Chl a (Fig. 1a, b). Addi-
tion of Trp again stimulated IAA levels, the endogenous
levels to a greater extent than the release (Fig. 2a, b),
and those of the symbiotic Nostoc more than those of
the free-living strain.
Chemical identification of IAA by GC-MS
To unequivocally demonstrate the chemical authenticity
of IAA in cyanobacteria, GC-MS was used. Represen-
tative mass chromatograms of two samples for the two
cyanobacteria are shown in Fig. 3. The true identity of
IAA in the cell extracts was hereby verified. This was
also demonstrated for eight additional samples repre-
senting the two Nostoc strains, analysed at various time
points and under different growth conditions (data not
shown).
A comparison of IAA concentrations obtained using
the ELISA immunoassay and the chemical GC-MS an-
alyses is shown in Table 3. A high positive correlation
between the two methods was observed. However, the
GC-MS method consistently demonstrated higher
endogenous as well as exogenous IAA concentrations,
irrespective of Nostoc strain examined or time expired.
As previously, addition of Trp stimulated both accu-
mulation and release of IAA in the two strains and this
was particularly evident using GC-MS. The latter assay
revealed about 2-fold higher IAA quantities than the
ELISA test (Table 3). After 2 weeks in the presence of
Trp, the endogenous levels of IAA amounted to 67 and
52 pmol per mg Chl a for the free-living and the sym-
biotic strains, respectively.
Identification of genes with homology
to IAA biosynthetic genes
A number of bacterial genes involved in the biosynthesis
of IAA have been identified and characterised. This to-
gether with the availability of several complete cyano-
bacterial genomes provoked us to search for genes
encoding homologues of bacterial and plant proteins
involved in IAA synthesis. However, a similarity search
for genes encoding enzymes of the major indole-3-
acetamide pathway, tryptophan 2-monooxygenases
(iaaM) and indole-3-acetamide hydrolases (iaaH; Patten
and Glick 1996) in Pseudomonas syringae and Agro-
bacterium vitis (GenBank accession numbers M11035,
Fig. 1 Accumulation (a) and release (b) of IAA by a free-living and
a symbiotically competent Nostoc, as evidenced by ELISA. The
values represent the mean (±SD) from two independent experi-
ments with three replicates each
233

M35690, AF061780, AF039169), gave no positive result.
This was the case in the three cyanobacterial genomes
tested: the unicellular Synechocysis sp. PCC 6803, the
free-living filamentous Nostoc/Anabaena sp. PCC 7120
and the symbiotically competent Nostoc punctiforme
ATCC 29133 (=Nostoc PCC 73102). Similarly, nega-
tive results were obtained for the P. syringae gene
encoding IAA-lysine synthetase (iaaL), converting IAA
to a less bioactive form (Roberto et al. 1990; Glickmann
et al. 1998). However, the genome of the IAA-releasing
N. punctiforme ATCC 29133 (Nostoc PCC 73102; see
Tables 1, 2) contains a gene coding for a predicted thi-
amine pyrophosphate-requiring enzyme, TPP-enzyme.
This 558-amino-acid-long protein shows similarities to
indole-3-pyruvate decarboxylases, encoded by the ipdC
gene, from Erwinia herbicola (GenBank accession num-
ber AAB06571), Enterobacter cloacae (BAA14242) and
P. putida (AAG00523). IpdC catalyses the conversion of
indole-3-pyruvate to indole-3-acetaldehyde (Koga 1995).
The amino acid identity values ranged from 41% to 43%
between IpdC from these bacteria and the homologue in
N. punctiforme (Nostoc PCC 73102). Comparison of
deduced amino acid sequences of IpdC of Enterobacter
cloacae with that of Azospirillum brasilense gave a 43%
homology (Costacurta et al. 1994). A full-length de-
duced amino acid sequence alignment of the putative
cyanobacterial and the bacterial IpdCs is presented in
Fig. 4. The alignment convincingly demonstrates that
the degree of homology between the predicted TPP-en-
zyme from N. punctiforme and IpdC from Enterobacter
cloacae, Erwinia herbicola and P. putida is higher than
the degree of homology between these bacteria and IpdC
from A. brasilense. Moreover, all amino acid residues
involved in the formation of the active site of IpdCs
(Koga 1995) are found in the conserved positions in the
sequence from Nostoc. This suggests that the cyano-
bacterial TPP-enzyme is contained within the bacterial
radiation of IpdCs. BLAST searches for the homologues
of this TPP-enzyme in public databases gave best scores
to the known and putative IpdCs (data not shown). In
addition, the genome of N. punctiforme contains several
copies of the aspartate aminotransferases (EC 2.6.1.1.)
Fig. 2 Accumulation (a) and release (b) of IAA by a free-living and
a symbiotically competent Nostoc in the presence of the putative
IAA precursor tryptophan (500 mg l
–1
), as evidenced by ELISA.
The values represent the mean (±SD) of two independent
experiments with three replicates each
Fig. 3 GC-MS identification of
endogenous IAA in the free-
living Nostoc 268 (in absence of
tryptophan; a, b) and in the
symbiotic isolate Nostoc
PCC 9229 (in presence of
tryptophan; c, d), both assayed
after 2 weeks of cultivation.
The peaks at m/z 202.1050
represent the IAA-methyl trim-
ethylsilyl ester (a, c) and the
peaks at m/z 208.1250 are indi-
cative of [
13
C
6
]IAA-methyl
trimethylsilyl ester (b, d), where
the [
13
C
6
]IAA was used as
internal standard (IS)
234

required for the first step of this pathway converting
tryptophan into indole-3-pyruvate. In the genome of
Synechocystis sp. PCC 6803, the product of merR shows
a high degree of similarity to arylacetonitrilase from
Alcaligenes faecalis (34% identity) and nitrilase II from
Arabidopsis thaliana (36% identity), enzymes involved in
the indole-3-acetonitrile pathway of IAA synthesis
(Bartling et al. 1994; Kobayashi et al. 1993). A similar
open reading frame (ORF) was also found in the ge-
nome of unicellular Synechococcus WH8102 (data not
shown).
Presence of the ipdC homologue in cyanobacteria
To complement our similarity search for putative IAA-
biosynthesis enzymes using BLAST, Southern hybridi-
sation of total DNA from 11 strains of cyanobacteria
(ranging from unicellular to filamentous heterocystous;
see Table 1) was performed using the PCR amplified and
cloned fragment of the putative ipdC gene from Nostoc
PCC 73102. Four positive ipdC signals were detected in
DNA of the seven strains shown in Fig. 5: in Nostoc 268,
Nostoc PCC 73102, Nostoc PCC 9229, and Anabaena/
Nostoc azollae. Four additional strains were also tested:
Gloethece sp. PCC 6909, Plectonema sp. PCC 73110,
Nostoc/Anabaena PCC 7120 and Nostoc PCC 6720, but
no positive ipdC response was obtained (data not
shown). Hence, it is apparent that although the majority
of these cyanobacteria responded positively in the IAA-
ELISA test (Nostoc LBG1 was not tested; Table 2), only
4 of 11 showed a response to the heterologous ipdC
probe.
Discussion
Here we provide the first evidence for the capacity
among cyanobacteria to accumulate IAA endogenously
and to release the hormone. We also demonstrate the
presence of homologues of the ipdC gene in some, but
not in all of the strains tested, a gene of key importance
in IAA biosynthesis. First, the Salkowski dye showed
release of auxin-like substances in 21 of the 34 cyano-
bacterial strains screened. The authenticity of IAA in
cell extracts and culture supernatants was then verified
using the specific monoclonal IAA antibody on 10 of the
screened Nostoc strains, and finally the chemical identity
of the IAA accumulated and released by 2 of these
strains was demonstrated using GC-MS.
It is apparent that the Salkowski reagent is the least
sensitive of the three methods used as some of the Sal-
kowski-negative strains responded positively using EL-
ISA and GC-MS (compare Tables 1 and 2), while the
reverse was never the case. Moreover, there is a positive
correlation between the ELISA and the GC-MS IAA
levels. But the GC-MS analyses in general gave higher
IAA levels than did the IAA antibody–ELISA test, and
particularly so in the presence of Trp. The reason for
this discrepancy is not known but the GC-MS method is
considered to be the most accurate.
The IAA levels in cyanobacterial suspensions are
difficult to compare with those reported earlier for
bacteria and other photosynthetic organisms, such as
plants, cyanobacteria being strikingly dissimilar in their
appearance. For instance, the IAA production for bac-
teria is often expressed per number of cells or per ml
culture medium. The cyanobacterial cell volumes are
about two orders of magnitude larger than those of most
bacteria, which leads to different packing efficiencies,
and makes comparisons of IAA levels per number of
cells or per ml unsuitable. However, cyanobacterial
cultures and protoplast suspensions are quite compara-
ble in their characters. After 2 weeks of growth in the
presence of Trp, the GC-MS recorded endogenous IAA
levels for the two Nostoc strains, amounted to about 12
and 9 pg IAA per lg Chl a, respectively (Table 3). These
levels are comparable to the about 146 pg IAA per lg
Chl a recorded after 7 weeks for a tobacco protoplast
suspension (Sitbon et al. 2000).
Our data also indicate that cyanobacteria may be
able to convert exogenous Trp into IAA (Fig. 2,
Tables 2, 3). However, it is possible that Trp was used as
a source of nitrogen during the initial stages of cultiva-
tion as the increase in IAA concentrations due to Trp
was most obvious during the later stages of cultivation.
Table 3 Comparative analyses
of IAA levels in a free-living
and a symbiotic Nostoc strain in
the presence and absence of
Trp. Data obtained by ELISA
and by GC-MS. The values
represent the mean ± SD from
two independent experiments
with three replicates each. a
IAA accumulation, r IAA
release, n.d. IAA not detected
Cyanobacterial
strain
IAA a/r Trp added (+) Cultivation period IAA (pmol mg
–1
Chl a)
ELISA GC-MS
Nostoc 268 a 72 h 10.0±0.5 15.8
Nostoc 268 a 2 weeks 10.1±0.4 12.5
a
Nostoc 268 a + 2 weeks 30.9±0.8 67.0
Nostoc 268 r 72 h n.d. 0.3
Nostoc 268 r + 1 weeks 4.0±0.4 11.3
Nostoc 9229 a 72 h 5.1±0.6 8.9
Nostoc 9229 a 2 weeks 14.9±0.6 17.8
Nostoc 9229 a + 2 weeks 28.5±0.7 52.0
b
Nostoc 9229 r 72 h n.d. 0.7
Nostoc 9229 r + 1 weeks 14.1±0.3 31.8
a
From Fig. 3a, b
b
From Fig. 3c, d
235

As Trp inhibits both heterocyst development and
nitrogen fixation, while its analogue, 7-azatryptophan,
provokes heterocyst differentiation in free-living An-
abaena cylindrica (Pain et al. 2000, p. 147), it is of in-
terest to examine the role of Trp on cyanobacterial
performance in general.
The above-mentioned observations related to IAA
biosynthesis, together with the results of the gene/amino
acid similarity BLAST search, suggest that a major
pathway for the production of IAA, at least in some
cyanobacteria, may be via the indole-3-pyruvic acid
pathway. The key enzyme (indole-3-pyruvate decar-
boxylase) of this pathway is encoded by ipdC and our
analyses revealed that ipdC-like genes are present in
some Nostoc species. Recently, it has been shown that in
A. brasilense, IpdC may account for most of the IAA
produced, that IAA is synthesised in a cell-density de-
pendent manner, and that accumulated IAA acts as an
autoinducer of IAA biosynthesis (Van de Broek et al.
1999). A similar IAA-accumulation effect was noted in
older cultures of cyanobacteria (Figs. 1, 2). Comparative
analysis of the amino acid sequences of several bacterial
IpdC enzymes led us to conclude that the TPP-enzyme in
Nostoc may be IpdC. This, however, will now need to be
Fig. 4 Full-length amino acid sequence alignment of deduced
indole-3-pyruvate decarboxylases (IpdCs) from IAA-producing
bacteria and a predicted TPP-enzyme from the cyanobacterium
Nostoc punctiforme ATCC 29133 (PCC 73102). Bacteria included
were Azosprillum brasilense, Enterobacter cloacae, Erwinia herbicola
and Pseudomonas putida (GenBank accession numbers P51852,
BAA14242, AAB06571, AAG00523). Identical amino acids are
shown in red, conserved in blue and similar amino acids in green.
The active site is marked by a cross, conserved residues in the
vicinity of the active site by bold arrows, the putative Mg
2+
and
diphosphate-binding domain by a bold line, and W denotes the
conserved tryptophan residue (after Koga 1995)
Fig. 5 Southern-blot analysis of total DNA from seven Nostoc
strains probed with a fragment of the putative ipdC gene from
Nostoc PCC 73102. Lanes: 1, Nostoc 268; 2, Nostoc LBG1; 3,
Nostoc PCC 73102; 4, Nostoc PCC 9229; 5, Nostoc Pc; 6, Nostoc-
Chr 894; 7, Anabaena/Nostoc azollae. All except Nostoc 268 (f) are
symbiotic isolates (s). DNA markers are given to the left
236

verified, as will the existence of other possible pathways
for IAA production in cyanobacteria.
Cyanobacteria may respond developmentally to
changed phytohormone signals both when free-living
and when in planta. Cyanobacteria are morphologically
more complex than most prokaryotes and some are
able to form several cell types (Rippka et al. 1979; Fay
1992). Most of these cell types appear in a strict timely
fashion in compatible Nostoc spp. infecting Gunnera.
First, small-celled motile hormogonia develop and la-
ter, after host cell penetration, these revert back into
multi-heterocystous vegetative filaments (Adams 2000;
Rai et al. 2000). As it has also been shown that IAA
triggers differentiation of cyanobacterial hormogonia
(Bunt 1961), the effect of IAA (and biosynthetic pre-
cursors) on cyanobacterial development in general is
now being tested.
Symbiotic cyanobacteria, like other plant-interacting
prokaryotes, whether beneficial (Azospirillum and
Bradyrhizobium) or pathogenic (Erwinia and Agrobac-
terium), may use the capacity to produce and release this
phytohormone as a strategy to influence endogenous
plant hormone levels and thereby plant performance
(Glick et al. 1999). Several developmental events are
known to be triggered in both partners during estab-
lishment of cyanobacterial–plant symbiosis. During
infection of the mucilage-secreting stem glands of Gun-
nera (Bergman et al. 1992, 1996; Rasmussen et al. 1994),
compatible Nostoc strains stimulate mitotic activity in
host cells close to the site of cyanobacterial host cell
penetration (Johansson and Bergman 1992). Also,
cyanobacteria provoke growth of plant protrusions
ramifying through the mucilage-filled cavities infected
by cyanobacteria in bryophytes and Azolla (Rodgers
and Stewart 1977; Peters 1991). IAA is known to cause
cell wall changes in plants, to enhance cell permeability,
to cause swelling of exposed plant cells and to stimulate
the production of a second hormone, ethylene (Liu et al.
1982). It is now of interest to examine whether cyano-
bacteria also make use of their IAA-release to initiate
the development of the symbiotic plant tissues.
In conclusion, our data for the first time reveal that
free-living and, in particular, symbiotic cyanobacteria,
are able to accumulate and release the phytohormone
indole-3-acetic acid, and that exogenous Trp stimulates
these processes. The next target in our investigation is to
isolate the putative ipdC gene from cyanobacteria and to
follow its expression, as well as to elucidate IAA-bio-
synthesis pathways and the role of IAA in cyanobacte-
rial cell differentiation in and plant symbiosis.
Cyanobacteria with their complex life cycle and their
unique ability to simultaneously fix nitrogen and per-
form oxygenic photosynthesis appear to share the same
mechanism for accumulation of IAA with some other
plant-interacting bacteria as well as with plants. In view
of the fact that the progenitors of extant cyanobacteria
were ancestors of plastids, the possible evolutionary link
between cyanobacterial biosynthesis of IAA and that of
plants is another aspect worth examining.
Acknowledgements The Department of Forest Genetics and Plant
Physiology, Sweden University of Agricultural Sciences (Umea
˚
,
Sweden) is gratefully acknowledged for GC-MS analyses of IAA.
We also thank the Royal Swedish Academy of Sciences, the
Swedish Natural Science Research Council and the Forestry and
Agricultural Research Council for financial support.
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