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THE REGULATORY ROLE OF POLYAMINES ON THE
LIGHT-INDEPENDENT PROTOCHLOROPHYLLIDE /
CHLOROPHYLLIDE CONVERSION IN THE MUTANT C-2A? OF
Scenedesmus obliquus.  Nikolaos E. Ioannidis and
Kiriakos Kotzabasis Department of Biology,
University of Crete, 71409 Heraklion, Crete,
Greece
Chlorophyll biosynthesis is a vitally important
process for the biogenesis of the photosynthetic
apparatus and consequently for the development of
chloroplast. The pigment mutant C-2A? of the
unicellular green alga Scenedesmus obliquus,
behaves like the higher plants since in the
darkness at 30oC accumulates protochlorophyllide
(Pchlide) and traces of chlorophyll. The
conversion of Pchlide to chlorophyllide (Chlide)
and the synthesis of chlorophyll take place under
light conditions (light-dependent Pchlide/Chlide
conversion). Alternatively the reduction of
Pchlide to Chlide in the C-2A? cells is possible
also in complete darkness under a lower
incubation temperature (lt25oC) (light-independent
Pchlide/Chlide conversion) Oh-Hama T.,
Kotzabasis K. and Senger H. 1987. Chlorophyll
estimations and fluorescence induction
measurements evidence a light-independent
chlorophyll biosynthesis and a development of a
mature and functional photosynthetic apparatus.
In vitro experiments with isolated etioplast
membranes (prolamelar bodies / pro-thylakoids
from C-2A? cultures grown in darkness at 30oC)
which incubated for different time intervals in
darkness at 20oC, showed also an unequivocal
light-independent Pchlide/Chlide conversion. This
suggests that the temperature changes act
directly on the activity of the
Pchlide-oxidoreductase enzyme. In the present
work the regulative role of polyamines and
especially of the tetramine spermine on
light-independent Pchlide/Chlide conversion, in
vivo and in vitro is demonstrated. Exogenously
supplied polyamines (putrescine, spermidine and
spermine), as well as polyamine inhibitors 1,4
diaminobutanone, cyclohexylamine and
1,3-diaminopropane show that either enhance of
spermine concentration or reduction of putrescine
seems to retard the Pchlide conversion to Chlide,
thus preventing chlorophyll biosynthesis and
chloroplast development. Exogenously supplied
spermine (1.0 mM) significantly inhibits the
light-independent Pchlide/Chlide conversion and
blocks the development of the photosynthetic
apparatus in vivo. Inhibition of the
light-independent Pchlide/Chlide conversion was
demonstrated also in isolated etioplast membranes
(prolamelar bodies / prothy-lakoids). The
possible mechanisms of polyamine influence on the
regulation of the light-independent
Pchlide/Chlide conversion is discussed.
All the experimental procedures of this work
were performed in complete darkness. Cultures
were grown heterotrophically in darkness under
various temperatures for 24 h. Cultures of the
mutant C-2A? grown in relatively high
temperatures (30oC) showed only traces of
chlorophyll whereas cultures grown at 20oC
present a 7-fold higher chlorophyll content and
lower Pchlide amount compared to the
corresponding one of the 30oC culture (Fig1).
(nmole/mlPCV)
ml
Fig 5. A. Phenotype presentation of C-2A cells
grown in absolute darkness at 20oC with different
concentrations of exogeneously supplied spermine
(0-3mM) compared with the phenotype of a C2A?
culture grown at 30oC. B. Chlorophyll and
Proto-chlorophyllide content (nmole/ml PCV) of
the above mentioned cultures.
Fig 3. Pigment content of the C-2A? mutant grown
in darkness at 30oC with different exogeneously
supplied polyamines (final concentration 1mM),
their inhibitors (final concentration 1mM) and
combinations of both (Put, Spd, Spm, 1,4DB,
1,3DP, CHA, Put 1,4DB, Spd CHA and Spm
1,3DP). Put putrescine, Spd spermidine, Spm
spermine, 1,4 DB 1,4 diaminobutanone, 1,3DP 1,3
diaminopropane CHA cycloexylamine.
A
B
Fluorescence induction measurements according to
the JIP Test of Strasser Strasser (1995)
supported the above results and suggests that
exogenously applied spermine blocks efficiently
the functioning of the photosynthetic apparatus.
Great differences in pigment content were
observed in the case of spermine and putrescine.
Fig.3 shows clearly that spermine inhibits and
the inhibitor of spermine biosynthesis, induces
the Pchlide/Chlide conversion. A similar to the
spermine effect was produced also by the
inhibition of putrescine biosynthesis (1,4DB).
Spermine inhibits the light independent
Pchlide/Chlide conversion also in vitro (isolated
PLB/PT membranes) (Fig. 4).
Fig 1. A. Pchl(ide) and Chl(ide) level of C-2A?
cultures grown in absolute darkness at permissive
(20oC) and non-permissive (30oC) for greening
temperatures. B. Packed cells of C-2A? grown
heterotrophically in the darkness (20oC lower
30oC upper tube) after centrifugation at 1500g
for 5 min.
The responsible enzyme for the reduction of
protochlorophyllide to chlorophyllide
(chlorophyll precursor molecule) is POR
(protochlorophyllide oxido-reductase). In an
attempt to examine the chlorophyll biosynthesis
mechanism apart from cell nucleus and gene
expression thylakoid membranes were isolated from
cells grown at 30oC and subsequently incubated at
20oC in darkness for different time periods. The
results of Fig.2 showed clearly that Pchlide was
reduced to Chlide also in isolated membranes in
absolute darkness.
Fig 6. Flurorescence induction kinetics of C-2??
mutant incubated at 20oC in absolute darkness for
48h with different concentrations of exogenously
supplied spermine.
2h Spm
Table 1. The values of the different structural
and functional parameters provided by the
JIP-test (Strasser Strasser 1995) (FV/FM
maximum yield of photochemistry, ABS/RC
absorption per reaction center, ETo/RC electron
transport per reaction center, DIo/RC
dissipation energy per reaction center,
RC/CSoActive RCs per excited cross section,
PI(abs) RCs density per absorbion PI(csm) RCs
density per cross section.
Fig 4.A. Isolated PLB/PTs membranes from C-2A?
cultures grown at 30oC in darkness incubated in
the presence of 2mM spermine at 20oC. The
Pchl(ide) and Chl(ide) level is presented by
fluorescence emission spectra. Spectra were
recorded with a Perkin Elmer LS 50B
spectrofluorophotometer. (Blue control (0h),
green 2mM spermine (2h) red control (2h)) B.
The percentage of the Chl(ide) increase and
Pchl(ide) decrease.

In vivo and in vitro measurements of
protochlorophyllide reduction, demonstrate that
the decrease of temperature in the C-2A? mutant
of Scenedesmus obliquus specifically influences
the catalytic activity of the Light-independent
protochlorophyllide oxidoreductase. This effect
cannot be attributed to de novo synthesis of a
product since this light independent
Pchlide/Chlide conversion takes place also in
isolated membranes. In the present work it was
found that polyamines influence the
light-independent Pchl(ide)/Chl(ide) conversion
and especially tetramine Spm seems to play a
significant role. In vivo and in vitro (PLB/PTs
membranes) incubation with Spm at permissive for
greening temperatures (20oC) showed an inhibition
of Pchlide/Chlide conversion and a block of light
independent chloroplast development.
Fig 2. Isolated PLB/PTs membranes form C-2A?
grown at 30oC in darkness that were incubated at
permissive for greening temperature (20oC) for
different time intervals (blue 0h, red 2h and
green 4h) and the Pchl(ide) and Chl(ide) levels
are presented by the fluorescence emission
spectra (arbitrary units). The excitation
wavelength was 430nm. All measurements were taken
at 95 acetone.
Exogenously supplied Spm in various
con-centrations showed that relatively low
concentrations of Spm inhibit the light
independent chloroplast development. Fluorescence
induction measurements according to the JIP test
of Strasser Strasser (1995) supported the above
results and suggests that exogenously supplied
Spm blocks efficiently the functioning of the
photosynthetic apparatus.
In an attempt to influence the reduction of
protochlorophyllide, we supplied exogenous
polyamines as well as inhibitors of polyamine
biosynthesis on C-2A? mutant. Exogeneously
supplied polyamines appear to influence
chlorophyll content when their final
concentration in growth medium reaches 1mM and
the incubation time of the cultures was 48h (Fig
3).

• Relatively low temperatures in absolute darkness
  permit chloroplast development of the chlorotic
  mutant C-2A? of unicellular green alga
  Scenedesmus obliquus.
• Incubation of isolated PLB/PT in darkness at
  relatively low temperatures (20oC) showed
  conversion of protochlorophyllide to
  chlorophyllide.
• The level of polyamines regulate the light
  independent protochlorophyllide to chlorophyllide
  conversion.
• The tetramine spermine inhibits significantly in
  vivo and in vitro (isolated plastid membranes)
  Pchlide/Chlide conversion and the light
  independent chloroplast development.
• AKNOWLEDGMENTS We thank Lambros Panagis for the
  photography work, Anastasia D. Gazis for the
  power point work and Elena Navakoudis for her
  useful suggestions and comments on the
  manuscript.