Dormancy of cells and organisms – strategies for survival
and preservation
Cyanobacteria Dormancy Forms in an Aquatic environment
AKINETES
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•
•
•
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What are they?
Their ecological role
Formation
Photosynthesis
Germination
What are they?
Akinetes - Cyanobacteria Dormancy Forms
 Akinetes (from the Greek ` akinetos', meaning motionless) are differentiated thickwalled, resting cells produced by many strains of subsections IV (order Nostocales)
and V (order Stigonematales), usually as cultures approach stationary phase.
 They do not resemble the endospore structurally. They and are not heat resistant,
but resist to cold and desiccation. They are larger than vegetative cells and have
thickened extra cellular envelope
 Akinetes contain higher amounts of storage compounds: glycogen & proteins
(cyanophycin) and maintain low level of metabolic activities
 Phosphate deficiency, specific light conditions, UV, simple organic carbon source
and un-aerated conditions induce the formation of akinetes
Akinetes - Cyanobacteria Dormancy Forms
 Aakinetes maintain residual metabolic activities as shown by incorporation of 35S
into protein and lipid. They consumed 02 in the dark and evolved 02 in the light
 Essentially every vegetative cell in the filament can differentiate into an Akinete
 Akinetes germinate to produce new filaments under suitable conditions
 Akinetes provide cyanobacteria with a means of over-wintering and surviving dry
periods
The ecological role of Akinetes
Schematic summary of the cyanobacteria life cycle
(prototype for species of the order Nostocales)
From: Hense I & Beckmann A (2006) Ecological Modelling (in press)
Development and maturation of Akinetes of Aphanizomenon ovalisporum
Australian strain of Aphanizomenon ovalisporum
was kindly provided by Lindsay Hunt, Queensland
Health Scientific Services.
Development and maturation of Akinetes of Aphanizomenon ovalisporum
20mm
20mm
Young akinetes perform photosynthetic activity
at a similar rate as their adjacent vegetative cells
600
Fluorescence (rel.)
500
400
0
75 110
175 260
365 510 850 1250
300
200
100
0
0
2000
4000
6000
8000
10000
Time (ms)
800
Fluorescence (rel.)
700
600
0
75
110 175
260
365
510
850 1250
500
400
300
200
2000
4000
6000
8000
Time (ms)
10000
12000
Photosynthetic parameters derived from Microscope-PAM measurements
140
120
Control
rETR
100
80
60
Vegetative cells in akinete - induced cultures
40
20
Akinetes on filaments
0
0
200
400
600
800
1000
1200
1400
-2 -1
Light intensity (mmol photon m s )
Vegetative cells
from
exponentially
grown cultures
Vegetative cells
from akineteinduced cultures
Akinetes on
filament of
akinete-induced
cultures
rETRmax
120.0  2.9
39.6  1.9
28.3  1.3
Initial slope
(a)
0.241  0.010
0.237  0.028
0.225  0.030
Ik
345
115
87
R2
0.994
0.962
0.956
Despite measurable F0 values, only residual Fv was detected with photosynthetic yields ranging between 0.05 and 0.1
Photosynthetic yield of Aphanizomenon vegetative cells and akinetes
Maximal Photosynthetic quantum yield of vegetative cells and akinetes of Aphanizomenon ovalisporum measured
by Microscope-PAM. Measured samples were vegetative cells in exponentially grown cultures, vegetative cells
and akinetes in akinete induced cultures and akinets isolated from 6 weeks old akinete-induced culture.
Vegetative
Cells
Akinetes
exponentially
grown culture
0.575  0.057
N/A
6 days
0.553  0.026
0.560  0.036
12 days
0.550  0.034
0.403  0.170
20 days
0.417  0.068
0.370  0.050
28 days
0.316  0.051
0.212  0.062
6 weeks
N/A
0.067  0.035
Sample
Control
Akinete-induced
culture
Isolated akinetes
Fluorescence emission spectra and their de-convoluted component
bands of Aphanizomenon ovalisporum Akinetes and vegetative cells
Fluorescence emission spectra were measured with excitation at 435 nm
0.20
0.20
0.16
A
0.18
721
651
Fluorescence (normalized)
Fluorescence (normalized)
0.18
663
0.14
0.12
0.10
693
0.08
685
0.06
0.12
694
0.10
684
0.08
0.06
0.02
0.02
661
736
649
650
700
750
0.00
600
800
Residuals
0.010
Residuals
0.14
0.04
0.005
0.000
-0.005
-0.010
600
721
0.16
0.04
0.00
600
B
650
700
Wavelength (nm)
750
800
650
700
750
800
650
700
750
800
0.010
0.005
0.000
-0.005
-0.010
600
Wavelength (nm)
Parameters of the component bands of fluorescence spectra at 77K of A. ovalisporum. Data was
extracted from steady-state fluorescence emission spectra and their de-convoluted component bands.
Fluorescence emission spectra were measured with excitation at 440nm.
Exponentially grown culture
Isolated Akinetes
Band
No
ID
lpeak
(nm)
1
PC
649
0.022
13
4.2
651
0.139
20
37.1
2
APC
661
0.042
9
5.8
663
0.063
10
8.3
3
Core Antenna
(F685)
684
0.056
7
6.4
685
0.051
6
3.9
4
PSII (F695)
694
0.095
12
17.9
693
0.066
10
9.1
5
PSI (FPSI)
720
0.172
21
56.9
721
0.158
19
41.2
6
Vibration
736
0.029
20
8.8
731
0.005
5
0.3
Hight
(rel.)
Width
(nm)
Area
(%)
lpeak
(nm)
Hight
(rel.)
Width
(nm)
Area
(%)
Characteristics of the component bands originated from PSII
(F685 and F695) and PSI (FPSI) of fluorescence spectra at 77 K
F685a
(Area %)
F695 a
(Area %)
FPSI a
(Area %)
PSI/PSIIb
(relative Area)
Vegetative
7.8
22.1
70.1
2.3
Akinetes
7.2
16.7
76.1
3.2
Cell type
a) The values were expressed as percentage relative to the sum of three emission bands (F685+F695+FPSI)
b) PSI/PSII fluorescence ratio is calculated from FPSI/F695
Abandance of cellular proteins (A) and Immuno-identification of PSII and PSI
polypeptides (B) in isolated akinetes (1) and exponentially grown culture (2) of
Aphanizomenon
150
non fluoresce
100
fluoresce
50
0
120,000
+P -K
Akinetes/ml
90,000
60,000
30,000
0
0
8
14
Days
21
free
attached
relative abundance (%)
In vivo fluorescence of Aphanizomenon akinetes
In vivo fluorescence of Aphanizomenon:
characterization by confocal laser scanning microscopy
Exponentially grown culture
exp018 Movie.wmv
vegetative cells
akinete
260
260
240
240
220
220
200
200
180
180
160
160
140
140
120
120
100
600
100
600
650
700
750
650
Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5
700
750
In vivo fluorescence of Aphanizomenon:
characterization by confocal laser scanning microscopy
Akinete induced culture 2 weeks old
induced030 Movie.wmv
akinetes on filament
vegetative cells
free akinetes (bright)
free akinete (dark)
250
250
250
110
200
200
200
100
150
150
150
90
100
100
100
80
50
50
50
70
0
600
650
700
750
0
600
650
700
750
0
600
650
700
Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5
750
60
600
650
700
750
In vivo fluorescence of Aphanizomenon:
characterization by confocal laser scanning microscopy
Isolated akinetes (from 6 weeks old culture)
akinete005 Movie.wmv
akinetes (bright)
akinetes (dark)
180
160
35
30
30
25
akinetes (dark)
140
25
120
100
20
80
15
60
15
10
10
40
20
0
600
20
650
700
750
5
5
0
600
0
600
650
700
750
Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5
650
700
750
Summary
 K deficiency triggers akinete formation in a yet unexplained process.
 Young akinetes maintain photosynthetic capacity at a similar manner as found for
their adjacent vegetative cells in filaments grown in akinete-inducing medium.
 Mature akinetes maintain residual photosynthetic activity.
 Some components of the photosynthetic apparatus appear to remain intact in
akinetes.
 In mature akinetes PSI and PSII complexes are kept apparently at a slightly higher
molar ratio then in vegetative young cells (less PSII).
 The phycobilisome pool is reduced in akinetes and disattached from the core antenna
complexes.
Akinetes differentiation dormancy and germination –
many processes are yet to reveal
From: Hense I & Beckmann A (2006) Ecological Modelling (in press)
Collaborators and students
Prof John Beardall, Monash University
Sven Inhken
Diti Viner Muzini
Bina Kaplan
Ruth Kaplan-Levi
Merva Hadari
Formation of akinetes
100000
Apha. akinets formation in BG - K2HPO4 under
different light intensities
Dark
Low light
High light
Cont Light
Akinets ml
-1
80000
60000
40000
20000
0
0
7
14
Days
High light – 120 mmol photon m-2 s-1 12/12 L/D cycle
Low light – 15 mmol photon m-2 s-1 12/12 L/D cycle
Continuous light - 15 mmol photon m-2 s-1
21
Formation of akinetes
Apha. Akinetes in BG-K2HPO4 with and without 0.2mM KCl
40000
Akinetes ml
-1
30000
+K LL
-K LL
20000
+K HL
-K HL
10000
0
0
2
9
Days
30
Physiological features of akinetes – literature survey
Akinetes of Nostoc spongiaeforme and Nostoc punctiforme remain viable and do not
germinate in distilled water, unless transferred to cyanobacterial growth medium (Huber
1985).
Non-germinating akinetes maintain residual metabolic activities as shown by incorporation
of 35S into protein and lipid. They consumed 02 in the dark and evolved 02 in the light
(Thiel & Wolk, 1983).
The composition of the external akinete polysaccharide layer has a structure similar to that
found in the heterocysts
Cyanophycin is a nitrogenous reserve material abundant in akinetes (Herdman, 1987).
Akinetes have increased resistance to environmental stress of desiccation, cold and
lysozyme treatment as compared to vegetative cells.
Formation of akinetes
P limiting conditions induce the formation of akinetes in Aphanizomenon
0.4
Biomass (mg/ml)
P quotient (mg/mg)
40
30
-P
Control
20
10
0
Control
0.3
-P
0.2
0.1
0.0
0
5
10
15
20
25
30
35
0
40
5
10
Time (days)
20
25
30
35
40
25
30
35
40
Time (days)
20000
Akinetes (No/ml)
100000
Akinetes (No/ml)
15
Akinetes on filamemts (-P)
Free akinetes (-P)
Total akinetes (-P)
75000
50000
25000
Akinetes on filamemts (Cont)
Free akinetes (Cont)
Total akinetes (Cont)
15000
10000
5000
0
0
0
5
10
15
20
25
Time (days)
30
35
40
0
5
10
15
20
Time (days)