Dormancy of cells and organisms – strategies for survival and preservation Cyanobacteria Dormancy Forms in an Aquatic environment AKINETES What are they? Their ecological role Formation Photosynthesis Germination

 Akinetes (from the Greek ` akinetos', meaning motionless) are differentiated thick- walled, 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 What are they?

 Aakinetes maintain residual metabolic activities as shown by incorporation of 35 S into protein and lipid. They consumed 0 2 in the dark and evolved 0 2 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 Akinetes - Cyanobacteria Dormancy Forms

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.

20  m Development and maturation of Akinetes of Aphanizomenon ovalisporum

Young akinetes perform photosynthetic activity at a similar rate as their adjacent vegetative cells

Photosynthetic parameters derived from Microscope-PAM measurements Despite measurable F 0 values, only residual F v was detected with photosynthetic yields ranging between 0.05 and 0.1 Vegetative cells from exponentially grown cultures Vegetative cells from akinete- induced cultures Akinetes on filament of akinete-induced cultures rETR max    1.3 Initial slope (  )    IkIk R2R

Photosynthetic yield of Aphanizomenon vegetative cells and akinetes Sample Vegetative Cells Akinetes Control exponentially grown culture  N/A Akinete-induced culture 6 days   days   days   days   Isolated akinetes 6 weeksN/A  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.

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

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. Band No ID Exponentially grown cultureIsolated Akinetes peak (nm) Hight (rel.) Width (nm) Area (%) peak (nm) Hight (rel.) Width (nm) Area (%) 1PC APC Core Antenna (F685) PSII (F695) PSI (F PSI ) Vibration

Characteristics of the component bands originated from PSII (F685 and F695) and PSI (FPSI) of fluorescence spectra at 77 K a) The values were expressed as percentage relative to the sum of three emission bands (F 685 +F 695 +F PSI ) b) PSI/PSII fluorescence ratio is calculated from F PSI /F 695 Cell type F 685 a (Area %) F 695 a (Area %) F PSI a (Area %) PSI/PSII b (relative Area) Vegetative Akinetes

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

In vivo fluorescence of Aphanizomenon akinetes

In vivo fluorescence of Aphanizomenon: characterization by confocal laser scanning microscopy Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5 Exponentially grown culture

In vivo fluorescence of Aphanizomenon: characterization by confocal laser scanning microscopy Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5 Akinete induced culture 2 weeks old

In vivo fluorescence of Aphanizomenon: characterization by confocal laser scanning microscopy Fluorescence was excited with the 488 nm line of Kr-Ar laser of a Leica TCS- SP5 Isolated akinetes (from 6 weeks old culture)

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) Sensing & signal transduction Development & maturation Dormancy & reduced metabolism Sensing, signal transduction & regulation of germination

Collaborators and students Prof John Beardall, Monash University Sven Inhken Diti Viner Muzini Bina Kaplan Ruth Kaplan-Levi Merva Hadari

Formation of akinetes High light – 120  mol photon m -2 s -1 12/12 L/D cycle Low light – 15  mol photon m -2 s -1 12/12 L/D cycle Continuous light - 15  mol photon m -2 s -1

Formation of akinetes

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 35 S into protein and lipid. They consumed 0 2 in the dark and evolved 0 2 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. Physiological features of akinetes – literature survey

P limiting conditions induce the formation of akinetes in Aphanizomenon Formation of akinetes