Biochemical Society Transactions ( 1 993) 21
473s
Changes i n carotenoids with nitrogen stress i n the
cyanobacteriun Synechococcus PCC6301
BASANTI BISWAL, LYNDON J ROGERS and ARNOLD J SMITH
Department o f B i o c h e m i s t r y ,
U n i v e r s i t y o f Wales
Aberystwyth, P e n g l a i s , A b e r y s t w y t h , D y f e d SY23 3DD
The c y c l i c c a r o t e n o i d s a r e components o f t h e
p h o t o s y n t h e t i c p i g m e n t - p r o t e i n complexes, and t h r o u g h
close association w i t h chlorophyll are essential
f o r assembly o f t h e p h o t o s y n t h e t i c a p p a r a t u s [l].
Here t h e y a c t as a c c e s s o r y l i g h t - h a r v e s t i n g pigments,
p r o v i d e a p h o t o p r o t e c t i o n mechanism and p e r m i t
dissipation o f
excess e x c i t a t i o n e n e r g y u n d e r
c o n d i t i o n s o f s t r e s s [Z].
This l a s t r o l e i s
a t t r i b u t e d t o z e a x a n t h i n formed b y d e - e p o x i d a t i o n
o f violaxanthin i n the so-called xanthophyll cycle
i n h i g h e r p l a n t s and green algae, o r formed de-novo
v i a 6 - c a r o t e n e i n c y a n o b a c t e r i a [3].
I n c y a n o b a c t e r i a t h e p i g m e n t - p r o t e i n complexes
include the phycobiliproteins,
organised w i t h i n
membrane-attached p h y c o b i l i s o m e s ,
i n addition t o
chlorophyll-protein
complexes.
T h i s assembly i s
disrupted r e a d i l y during nitrogen starvation leading
t o substantial loss o f the phycobiliproteins within
24h o f d e p l e t i n g c u l t u r e s o f n i t r a t e , w i t h s m a l l e r
changes
in
chlorophyll
and
carotenoid
[4].
Supplementation o f t h e c u l t u r e s w i t h n i t r a t e w i l l
q u i c k l y reverse t h i s loss.
Concomitant w i t h o t h e r i n v e s t i g a t i o n s o f t h e
coordination
of
chromophore
and
apoprotein
biosynthesis
for
phycobiliprotein
assembly
in
Synechococcus PCC6301 we have a l s o l o o k e d a t changes
i n carotenoids i n c e l l s subjected t o n i t r a t e
s t a r v a t i o n and d u r i n g t h e i r subsequent r e c o v e r y .
I n f o r m a t i o n on such changes d u r i n g t h e i m p o s i t i o n
o f s t r e s s on p h o t o s y n t h e t i c organisms i s f r a g m e n t a r y
and o f t e n c o n t r a d i c t o r y .
The e f f e c t s o f n i t r a t e d e p l e t i o n i n terms o f
phycocyanin, c h l o r o p h y l l - a and c a r o t e n o i d c o m p o s i t i o n
o f t h e c e l l s was f o l l o w e d f o r 48h.
A comparable
nitrogen-depleted
c u l t u r e was t h e n supplemented
w i t h n i t r a t e and t h e r e s t o r a t i o n o f p i g m e n t a t i o n
followed over a s i m i l a r period.
The d e t a i l e d
methodology f o r t h e s e s t u d i e s w i l l be p r e s e n t e d
elsewhere. The d a t a was a n a l y s e d i n terms o f amounts,
p e r c u l t u r e volume and p e r g o f c e l l m a t e r i a l ; b o t h
d a t a s e t s a r e n e c e s s a r y s i n c e t h e c e l l wet w e i g h t
c o n t i n u e s t o i n c r e a s e even d u r i n g n i t r o g e n s t a r v a t i o n
due t o c o n t i n u i n g s y n t h e s i s o f p o l y s a c c h a r i d e 141.
I n t h e samples v i r t u a l l y a l l t h e c a r o t e n o i d
( a v . 97%) was made up o f 6-carotene and z e a x a n t h i n .
I n t h e s t a r t i n g c u l t u r e i n t h e l o g phase o f g r o w t h
6-carotene made up some 54% o f t h e c a r o t e n o i d , and
z e a x a n t h i n a b o u t 43%.
As c e l l s became n i t r o g e n
l i m i t e d t h e phycocyanin declined d r a m a t i c a l l y t o
31% o f t h e o r i g i n a l c o n t e n t on a wg m l - l c u l t u r e
b a s i s whereas c h l o r o p h y l l was r e t a i n e d and c a r o t e n o i d
synthesis continued,
i n c r e a s i n g b y 60% i n 48h.
T h i s l a t t e r change was accounted f o r b y an i n c r e a s e
i n z e a x a n t h i n w i t h B-carotene c o n t e n t d e c l i n i n g
only
slightly.
As
a
consequence,
in
the
n i t r o g e n - d e p l e t e d c u l t u r e s a - c a r o t e n e made up o n l y
25% o f t h e c a r o t e n o i d p r e s e n t , and z e a x a n t h i n 75%.
On
nitrate
replenishment
of
the
culture
z e a x a n t h i n was n o t produced f o r 36h, b y which t i m e
fi-carotene l e v e l had i n c r e a s e d some 4 - f o l d a l o n g s i d e
a s i m i l a r i n c r e a s e i n c h l o r o p h y l l and t h e f u l l
r e s t o r a t i o n o f phycocyanin; t h e r e l a t i v e p r o p o r t i o n s
o f t h e two c a r o t e n o i d s were t h o s e c h a r a c t e r i s t i c
o f the o r i g i n a l nitrogen-replete c e l l s (Fig.1).
Analyses
of
these
data
showed
that
notwithstanding the large fluctuations i n r e l a t i v e
pigment l e v e l s t h e r a t i o o f a-carotene t o c h l o r o p h y l l
remained m o r e - o r - l e s s c o n s t a n t t h r o u g h o u t a t 0.14
t s . 0 . 0.02
( 1 0 ) whereas
t h a t o f zeaxanthin t o
c h l o r o p h y l l v a r i e d f r o m 0.12 i n n i t r o g e n - r e p l e t e
Fig.1.
Changes i n t h e r e l a t i v e p r o p o r t i o n s o f t h e
c a r o t e n o i d s p r e s e n t i n Synechococcus 6301.
(A)
C e l l s were t r a n s f e r r e d t o a n i t r a t e - f r e e medium
a t z e r o t i m e and t h e r e a f t e r sampled a t 12h i n t e r v a l s .
(B) A t 48h t h e medium i n a s i m i l a r l y - t r e a t e d b u t
unsampled c u l t u r e was supplemented w i t h n i t r a t e
and t h e r e c o v e r y f r o m n i t r a t e s t a r v a t i o n was f o l l o w e d
o v e r a f u r t h e r 48h.
C a r o t e n o i d s were e x t r a c t e d
i n 100% acetone and r e l a t i v e amounts e v a l u a t e d f r o m
peak a r e a s (A436nm) f o l l o w i n g s e p a r a t i o n o f t h e
c a r o t e n o i d s and c h l o r o p h y l l - a b y HPLC on an ODS
non-endcapped r e v e r s e phase c o l umn ( L i c h r o s p h e r e
100 RP-18) u s i n g a g r a d i e n t o f 0-100% e t h y l a c e t a t e
i n acetonitrile:
w a t e r (9:1, b y v o l . ) o v e r 20 m i n
w i t h a 1.0 m l m i n - 1 f l o w r a t e .
The system w i l l
separate zeaxanthin from l u t e i n .
This
c u l t u r e s xco 0.34 i n n i t r o g e n s t a r v a t i o n .
suggests t h e r a t i o o f 6 - c a r o t e n e t o c h l o r o p h y l l
may be r e g u l a t e d b y t h e i r d u a l r o l e s i n t h e assembly
o f t h e p h o t o s y n t h e t i c complexes, whereas c h a n n e l l i n g
in
biosynthesis
to
yield
zeaxanthin
as
the
predominant c a r o t e n o i d i n n i t r o g e n - s t r e s s e d c e l l s
is
consistent with
its
protective role.
In
Synechococcus 6301
the
photosynthetic
lamellae
contain e s s e n t i a l l y a l l t h e 6-carotene
whereas
t h e g r e a t e r p a r t o f t h e z e a x a n t h i n i s found i n t h e
c y t o p l a s m i c membrane [ 51.
Our d a t a and c o n c l u s i o n s c o n t r a s t i n some
r e s p e c t s t o s t u d i e s o f Synechococcus 6301
where
phycocyanin d e g r a d a t i o n d u r i n g n i t r a t e s t a r v a t i o n
correlated
with
the
loss
of
membrane-bound
c a r o t e n o i d s [ 6 ] and o f Phcimidium laminosus where
a decrease i n t h e r a t i o o f 6-carotene t o n o s t o x a n t h i n
during
nitrate
s t a r v a t i o n was
interpreted
as
s u g g e s t i n g t h a t t h e p h o t o s y n t h e t i c membranes were
more a f f e c t e d t h a n t h e c y t o p l a s m i c membrane [7].
D r B i s w a l was i n r e c e i p t o f a Commonwealth Academic
S t a f f F e l l o w s h i p t o UWA f r o m Sambalpur U n i v e r s i t y ,
Jyotivihar, India.
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