Polyamines influence maturation in reproductive structures of

Polyamines influence maturation in reproductive structures of

POLYAMINES INFLUENCE hIATUR4TION IN REPRODUCTIVE STRUCTURES OF
GRACILARIA CORNEA (GRACILARLALES,RHODOPHYTA) '
1
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murno w ~ ~ ~ r u ~ ~ - u n u s ~ e p z
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Centro de Investigación y de Estudios .4\anzados del IPh' - Unidad Mérida. A.P. 73 Cordemex Mérida, Yucatán. México
Pilar García-Jimtha,Fernando Manán
Departamento de Biología, Universidad de Las Palmas de Gran Canana, Campus Universitario de Tafita C.P. 35017,
Las Palmas de Gran Canana, Esparia
nn.&-1 1RLrVi"L
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- 2
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U U I ( i ' I C
Centro de Investigación y de Estudios Avanzados del IPN - Unidad Ménda, A.P. 73 Cordernex Mérida,Yucatán. México
and
Rafael Robaina
Departamento de Biología, Universidad de Las Palmas de Gran Canaria, Campuk Universitario de Tafira C.P. 35017.
Las Palmas de Gran Canana, España
Naturally occurring levels of putr-e,
spermidine,
and spermine were analyzed in female gametophyte
(nonfertiiizecij anci reprociuctive tissues (cystocarpsj
at two different stages of development in the marine
red algae Gracilarin. cornea J. Agardh. Endogenous
polyamine levels changed at differential stages of
cystocarp maturation. Highest p.yamine values were
founci on iissue from &e earj post-feroiization -e,
decrease as the cystocarp matured. Incubation experiments revealed that exozenous polyamines induced qstocarp maturation and promoted carpospofe
liberation, developing ceil masses witbin 4 to 7 days m
treatments with spermine. This is the first report on
the effect of polyamines on cystocarp maturation in
marine algae.
K ~ Jindex w d : carpospores; cell mas; cystocarp
. mahuation; Gmalwuz annsa; poiyamines; putrescine;
spermidine; spermine
Abbratiaiiom: PAs, polyamines; PES, Provasolienriched . seawater; PUT, putrescine; SPD, spermidine; SPM, spermine; TCA, h-ichloroacetic acid
Polyamines (PAs), spermidine (SPD), spermine
(SPM), and their diamine obligate precursor putrescine
(PUT) are small aliphatic arnines that are ubiquitous in
al1 plant cells. They have been labeled as a new class of
growh substances and a type of plant growth regulator
or hormonal second messenger (Galston and KaurSawhney i999, Lee and Shu i992, liburcio et ai.
1993, Gaspar et al. 1997, Scoccianti et al. 2000, Tassoni et al. 2000). The polycationic nature of PAs resulted in covalent bonds with macromolecules and
thus affected the synthesis and activity of hose mole'Received 29 October 2001. Accepted 12 September 2002.
2Author for correspondence: e-mail [email protected]~estav.mx
cules. As other plant growth regulators, PAs also react
with soluble substances forming conjugates (i.e. phenoiic acicisj. As a resuit, they can De founa in severai
tissue fractions, such as free acid soluble and bound
acid soluble, which must be hydrolyzed to be quantified, and bound acid insoluble, associated tightly to
the tissue whose presence are also revealed after hydroiization. Tiie roie of each of hese kactions remains elusive, bu[ their possible role as a reservoir of
PAs that control the endogenous levels of active and
free PAs, as occurs with other plant hormone conjugates, has been suggested (Martin-Tanguy 1997, Pandey et al. 2000).
PAs are important for developmental proceses such
as pollen m a m t i o n , germination, and flower develop
ment (Smith 1985, Tomgiani et al. 1987,
et al.
1988, Evans and Malmberg 1989). Extensive studies
süPPOri their ro!e iii %e mollU!aho-il of a ~ R e af
q
physiological processes, such as stabilization of cell
membranes (Schubert et al. 1983, Roberts et al. 1986,
Kaur-Sawhney and Applewhite 1993), stress response
(Flores 1990, hurisiano et al. 1993, Das e t al. 1995, Galstm e: d. 1997, r-I
et d. 2MG), a ~ !senescence
(Slocum et al. 1984, Evans and Malmberg 1989, Rey et
al. 1994, Del Duca et al. 2000). Nevertheless, their
precise mode of action is yet to be understood, as is
the case for other plant hormones (Walden et al.
1997). In recent years the interest in PA research has
increased tremendously and is now being applied to
study important agronomic and horticultura1 crops
(Ka~am199'7).
In marine algae, PAs have been studied in relation
to their occurrence within different alga1 groups
(Hamana and Matsuzaki 1982) and their involvement
in cell division (Cohen et al. 1984). Inforrnation on
endogenous levels, uptake, and transport within the
thallus has been reported for Ulua rigida (Baldini et al.
1994). Lee (1998) reported accumulation of PUT and
SPD rclated to lethal hyposaline stress in severa1 spccies of intertidal marine macroalgae. Most recently,
PAs have been used for studying in vih-o regulatory
events of sporeling morphogenesis for Gruteiuupia
d q p h o r a (García-Jiménez et al. 1998, Manán et al.
2000a). To our knowledge, no single study has monitored endogenous levels of P h during maturation of
reproductive structures in marine algae nor the effect
ef exegefi^US P.AS en i& &ue!enrn~ntal
c t a u ~ 1 c rer"'-"-'
--a-'
ported for terrestrial plants (MTaldenet al. 1997).
Reproduction in red algae occurs by both sexual
and asexual processes through the release of sexually
or asexually produced spores that are borne in sporanga. The classification of red alga1 sporangia is best
based on morphological attributes (Guiry 1995). Fertilization invoives the fusion of a nonflaiellated
male
"
gamete, the spermatium, with a sessile egg-containing
cell, the carpogonium on the female gametangial
thalli. Mter fertilization, the zygote is not released directly but is retained as a parasite on the female gametangial thalli where it develops into a third generation, (he carposporophyte. After fertilization, the
carposporophyte of the red algae may appear to be
-
.
.
~.L-L. 2
r
.
~ u i r o u i2i u- 3c uLuy,
g-d.~rit.~opriyuc
ussuc, ioixiiiig
-
A i i i ~ ~ r u
scopically visible reproductive structure, the cystocarp.
Cystocarps may appear as smaii dark spon (1-5 mm diameter) embedded in the thalli as small domoid rvarts
on the surface of branches. The qstocarp may eventually
release the carpospores, which seme as the agents of recruitment for the tetrasporophyte generation, a tetraspore producing phase. Tetraipres originate male
and female garnetangial thaiii, completing the life cycle.
In this study we analyzed endogenous levels of PAs
at two ciiierent stages of cystocarp beveiopment, eariy
post-fertilization (immature stage) and mature cystocarp
in the carpospornphytic p h z e , of the o d macroalgae
Gracilana c o n wJ. Agardh. Furthermore, in vitro expenments were conducted to examine the effect of exogenous PAs o n cystocarp maturation a n d carpospore
development
MATERIALS AND METHODS
Planf
Arnong the species of G ~ ~ l a r in
i uthe westem
Atlantic Ocean, the alga known as G. c m a is one of the most
common and easily recognized. It is widely disuibuted along
tropical and s ~ i h t q K -cl o a - ;md is i~sedLocally a a snurcp of
agar and as a food supplement (Robledo 1998). In G. cornea cystocarps are scattered over at least the upper half of the thallus
and protrude only moderar+. being small relative to h e thallus
diameter, about 2 mrn high and 4 mm wide. From the time of
carposporophyte initiation until full maturity, the cystocarp
height remains uniform, with the principal expansion in width.
The ontogeny of these structures has been reponed in detail by
Fredencq and Norris (1985).
Female gametophpes without fertilized carpogonia and carposporophytic thallus of G.c m e a were collecred from a natural
bed located at Dzilam de Bravo, Yucatán, México. After collection, female garnetophytes and carposporophytes were cleaned,
bmshed, and nnsed several times in sterile seawater to eliminate epiphytes and substrate. The matenal was maintained 1
week in sterile seawater with Provasoli-enriched seawater (PES)
media (Provasoli 1968) at 26" C. 40 pmol photons-m-"S-', and
an 8:16-h 1ight:dark photoperiod.
In the [weseiit stiidy two stages ofcarpospuropityte devciol~
ment were identified and separated based ori initial post-fertilia t i o n events described by Hommerwid ancl Fredei-icq (19%)
Sor rhe order Gracilariales. The first cell derivatives of the fertilized carpogonium give rise directly to gonimoblast filaments
and grow over the outermost cortical cells, eventually fusing
with them. Additional fusion with neighboring gametophytic
,--Il1-..A
.+,
.L-C,.-...:-,.Cm 1
1-l.-2
C..-:..--11
:A,.-a:C-A
CLLIJ
I C ~ ULV U ~ C
I W I I I I ~ L I V I L V L a iaisr i u i , c c i i i i ~ i u i ~
i cii
iuriiriiicu
by a dark spot on the carposporophytic thallus (immarure
stage). After the gonimoblast have expanded, filling die cysro-
----
-..caiy
+L.-..
-".;h.
C ~ V ~ L JL ,I I L ~
:-:.:-.r i i n u a t z
&..-.--L.--
U i a n L L l i L a
A$
w
i
,-..-,."-----:"
--A
"l?-
L P I ~ V D ~ V I P L ~ aaju
S I ~ ata\,
produce tubular nutntive cells that fuse with pericarp cells or
with cells in the floor of the cystocarp, developing a protuber.,..+*:**..--"A
L1.LL
U
.
,
"
,
.
1--,4:-e.,
allu L L a U I I I ,~.L
,
.c
*
.
-..
C,'JL"C'%ly
-..*...-..;--
I . l a L U I a I I " I 1
.he.
;--.;A-..,
,,
Lamal .
a LVICILIIL L
V I
the presence of the ostiole (mature cystocarp). Endogenous
levels of PAs were analyzed in female gametophyre without fertilized carpogonia and in the two crjtocarp developmental
stages described above, easily recognized stages in the G. cumea
life cycle (Fig. 1).
To evaluate the effect of PAs on cystocarp maturation, 10 mature qstocarps at each stage of development were incubated
separately in Peui dishes for 24 h in PES and PES plus common
PAs at
M (PES + PLT,PES + SPD, and PES + SPM), which
were maintained under 26" C, 40 pmol photon~-rn-~.s-',
and an
8:lGh Iighcdark photoperiod, the best conditions for carpospore
LL-Lic..7-;-~~
I..xP,--.;
..-A
R
~
L
I
D
,
L
i
aoa)
P ~ - ~ c ~ - w . , ~
\"U""'""-L,"""c6U'
released from the above expenrnent were counted in a counting
chamber and maintained for 22 days in their original media un&y
C , 2fi
phGtGnT.m-2.s-l, 2nd an Y:!$h T ; r r h r . A q r l .
uauL.-a "
photoperiod to follow carpospore developrnent.
PA amiysis. Free (as free cations), bound acidsoluble, and
bound acid-insoluble (acid-insoluble forms bound covalently to
macromolecules and cell walls) PAs (PUT, SPD, and SPM) were
extracted in cold 5% trichloroacetic acid (TC4).P.45 were anaIyzed from 70 to 100 tissue fragments (explants 6 mm lengthi
from nonfertiiized female gametophyric and G. -a
qstocarps
at different stages of maturation in a 1:10 ratio of algae material
(g Frerh weigh!!:5% T U (EL). The s l u q iias cer.tRfcged at
1500gfor 15 min, and the supematant was divided in half for
the analysis of free T U soluble and bound TCA soluble, whereas
~ d&e ma!j~k uf h m d TCA-ksu!iih!e P.k
h e @!et m l l ~ fur
(Manán et al. 2000a). Bound T'U soluble (200 pL) and bound
TCA insoluble (pellet) were hydrolyzed in separated sealed \~ials
Gth 300 p,L nf 12 M HC! fer 20 h ar !!M0
C to o!eze h e b l m d
fraction. After hydrolysis the samples were fiftered, dried to e v a p
m e the remaining HC1, and redissolved in 300 pL of 5% TC4 for
dansylation (24 h, 60" C) .
Danrylalim of P h f m TLC. The procedure for danqlation and
quantification of PAs used a I W K L sample mked with 100 pL of
a saturated solution of Nazco, and 200 pL of dansvl chlonde
(5 mg-mL-' acetone). The dansylation reaction lasted for 10
min at 70" C. Aftenvard, 50 FL of an aqueous proline solution
(100 mg-mL-') was added to react for 30 min in darkness with
the excess dansyl chloride. When the reaction was completed,
500 pL of toluene was mixed with che sample, shaken, and left
unti1 The o w i c md aq~ueousphzwc. weo p z m e d The orpnic
phase, containing the danql-PAs, was dried in a heat-speed Lacuum
and the residual nwi dissolved in 600 pL of acetone. The dansyl-PAs
were separated blth TLC on 7.5 X ?..?cm plates (F15(Kl/IS2234.
Schleicher & Scuell, Keene, NH, USA). The mobile pliase was a
chloroform/triethylamine mixture (51, v/v). The plates ivere revealed under UV lighr (254 nm). and the bands refereed to thc
dansylated standards of PUT. SPD, and SPAl (Sigma. St. Louis,
MO. USA). These bands were scraped off the plate and redissolved
with 800 pL acetone. The samples were shaken and centrifuged
at 6000gfor 3 rnin, and 500 p L of this solution has quanrified ar
365 nm (excitation) and 510 nm (emission) with a high-resolu~oii
spectrofluorometer (SFM 25, Kontron Instruments, Zurich, Swilzerland). Three replicates were quantified for each sample.
Histolog. Mature Ttocarps ( n = 3) from h e incubation e*periment were fxed with 5% glutaraldehyde in lo-' M sodium cacodylate buffer containing 0.25 M sucrose (pH 7.4) for 4 h at room
ternperature (García-Jiménez et d. 1998). This %-as followed b v
U""'"""
""U
'"""Y"
Lddd,.
ULLly"Jy"lcJ
S8
Y
/
Tetraspores
-
in sini
fecundation
Female
Carposporophyte with
inmature cystocarp
Gametophytes
"1.
1
I
Tetrasporophyte
-
\
Carposporophyte with
mature cystocarp
4
carpospores
2n
F'IG. 1. Tnphasic life cycle of GraaIaria unnea, showing the extemal morphology for the two stages of qstocarp maturation studied
within the carposprophytic phase.
four washings (one each for 30 min) in the sane buffer containing
sucrose in descendant concentrations (0.25, 0.12,O.M. and O M)
and embedded in glycol methac~iate(HistoresinTM,
Leica, Nuss
loch, Germany) (Gemds and Smid 1983). Serial sectionc 5mm
thick were cut on a Reichertjung 2050 rnicrotome and stained for
the oDsenation of generai morphoiogy with toiuiciine biue -sekos 1983).
Staiirlical amlysis. Statistical analysis was performed with
Statgraph'ics Pius for Windows, version 2.1 (hlanugistics, Rockviile, MD,USA). The data were analyzed by &teststo find out
differences between endogenous PA levels at different develop
mental stages of the cystocarp and to determine differences
Detween íemaie garnetophyte and each stage oí maturation oí
the qstocarp.
TABLE1. Endogenous P.4 contents (wg.gl fresh weight) reported in other studies for manne macrophytes and the present study
for Graciiaria m a .
SPM
SPD
Free
BS
BI
Free
C b a jarciala Delilea
Chaetmmpha crma
(C. Agardh) Kützing"
Valoniopsispachynema
(Martens) Bwgesena
Uiciyaiu dic;cuiumu
(Hudson) Lamourouxb
Ceizdium cananksls (Gmnow:)
c
,---I.-b
.,L"aIILUQI,I"Oi
Graleloupia doryphora
(Montagne) Howeb
Cymodocea nodosa
(Ucria) AschenonC
Gracilaná c m a J . Agardhd
Female gametophyte
Imrnature cystocarp
Mature cystocarp
'Lee 1998.
Manán et al. 2000a.
Marián et al. 2000b.
ThlSs:U&Í.
-, Not wported; BS, bound acid soluble; BI, bound acid insoluble.
d
BS
BI
Free
BS
BI
RESULTS
Endogenous h e I s of PAs. PA analysis revealed that
PUT was the predominant amine, followed by SPM
and SPD. Tne bound acici-insoiubie forms of PAs were
more abundant, followed by the free and bound acidsoluble forms. This was more evident for PUT, where
a 10-fold difference was observed. PA contents were in
range with those obtained for other algae (Table 1).
1 ne ciifferent forms of PAs significantiy increased
(P < 0.05) from female gametophyte (nonfertilized carpogonia) to immature cystocarp (early post-ferrilization),
except for PUT free and bound acid soluble and SPD
bound acid soluble (Fig. 2). Mature cystocarps had
iower ieveis of P ü i (fi-ee, -bound acid soiubie, and 'bound
acid insoluble), SPD (bound acid soluble), and SPM
(bound acid soluble) than female gametophytes.
-.
0
m
m
Female Gametophyte
Immature Cystoarp
Mature Cystocarp
PUT
The concentrations OS al1 three PAs were higher in
irnrnature cystocarps (early post-fertilization stage) than
in mature cystocarps. PA analysis for imrnature cystoc q s re~Jeded
~ h , &e
~ : PCT con:efi: (haAr,íj a ~ inso!üd
ble) was highest, decreasing for mature cystocarps; this
was also observed for al1 forms of SPD and SPM.
PA incubation experiments. Histological obsemtions
on mature cystocarps after PA incubation showed the
~
4
T
LALLLL
~
AI
r t P V ~ C T P - T \ . .D
~A . .
vr
r
m
A-
~ ~ v g ~ i i v u auii
-C
iiiawiauuii vi
cya~u~aip.
-..C..-&:--
Exogenous addition of PAs promoted high disorganization of carposporangial branches inside cystocarps.
This disorganization was more evident for SPM (Fig.
3, A and B).
T--..LqL--C-*+----.:D A - -----+-A
--------,.
utcuuauuti UI ~yx-ya
111 1 N ~ I U I I I U L C Uu u p ~ y u ~ c
release; the number of carpospores liberated increased at least 40% when compared with liberation
in PES (Table 2). A higher number of carpospores
were liberated in SPM, with a Iower output in PUS
and SPD. Dunng cultivation in media with PAs, the
growth and development of carposporelings was enhanced. In PES, successive ceilular divisions were evident until the formation of one main axis after 21 days
of release, whereas PAs increased cell division dunng
carpospore development, produced cell masses, and induced morphogenesis. In PUT, cell masses were
forrned between 12 and 15 days after release. A most
evident effect on cell division was observed in SPM,
where cell masses were obtained after 4-7 days. Differences in axes number and size were also evident for
cell masses obtained in treatments with PAs. In PUT
axes were larger, whereas in SPD and SPM more than
one axis developed (Fig. 3, G F ) .
DISCUSSION
2501
SPM
a c
a
.
Free
.
BS
BI
Frc. 2. PA contents at various stages of cystocxp develop
ment in the femaie gametophyte of Graaláa unnea. PA contents
in nonfenilized female gametophyte are 100%. BS, bound acid
soluble; BI, bound acid insoluble. Sarne letter indicates significant
diíferences (P < 0.05) between female gametophyte, immature
cystocarp, and mature cystocarp.
PA pattems differ greatly among algae not only in
the amount of PUT. SPD, and SPM, but also in the
amount of free, bound acid-soluble, and bound acidinsoluble fractions. Endogenous levels of PAs in G.
c m a revealed a higher amount of PUT. The presente of PAs has been reported for other algae, with
PUT beiny the most abundant in green algae (Baldini
et al. 1994, Lee 1998). SPM in G. conea was found in
higher amounts than SPD, contrary to trace levels of
SPM found in green algae (Hamana and Matsuzaki
1982). Nevertheless, PA levels in the nonreproductive
(female gametophyte) and reproductive tissue (cystocarps) of G. c m e a are within the range descnbed earlier for other algae.
W na~uraiincrease in enciogenous ieveis o i IiHs ciuring cystocarp development at early post-fertilization
events was evident when compared with PAs found in female thaili without fertilized carpogonia. Our results
suggest that endogenous PAs levels in reproductive tissue of S. unnea are associated with differentiai stages of
cystocarp maturation. Carposporophyte evolution has
involved the progressive modification of garnetophytic
tissues and the structural and functional compartmentalization of the cystocarp. Hornrnersand and Fredencq
(iW5j recognized uiree cornpartments of the cystocarp:
the outer photosynthetic tissues, the modiied inner ga-
POLYAMINES INFLUENCE CYSTOCAKP MATURATION
F I ~ 3.
. Cystocarp and carpospore developrnent in
GraalaTin carnea (A) Longitudinal section of rnamre cystocarp incubated in PES and [B) in PES plus SPM
M:
Note differences in the arrangernent of carpogonial
branches (arrows). ( C ) Ce11 masses originated from carpospores in PES plus SPM
M, 7 days old and (D) carpospore development in PES, 22 d a old.
~ Axes develop
ment after 22 days in (E) PUT
M and (F) SPM
M. Scale bar, 0.2 rnm.
T A I ~ I .9I . Oarposporc release of (;racilrrnrr r o r n ~ rinaliirc
Vtocarps after incubation for 24 h in PES and PES plus common
PAsat 10-".
gametophyte without fertilized carpogonia) to early postfertilization event (immature cystocarp), most probably
due to increased cell division. Addition of exogenous PAs
dunng the final stages of carposporophyte development
promoted carpospore release, cell division, and morphogenesis. Fut-ther studies are needed to understand the
mode of action of PAs and other plant horrnones.
aSignificant differences ( P < 0.05) in carpospore release
between PES media and PES plus PAs.
SupprrPd hy the g m m m e n t of h e C h q k h & (PITCK)1/129).
COKACYT 3605CB and by IFS grant A/2414/2. k GuzmánUnóstegui is grateful for a p
t from AECI and to a PhD Scholar-
metophytic tissues that process and store the metabolites
of photoqnthesis, and the developing carposporophyte.
Increased nutritional dependency on the gametophyte is evident in the fict that carpospo~ophyte
growth is not a continuous process; it proceeds in
stages, with penods of rapid growth followed by interMIS of slow growth or apparent inactivity dunng
which the pattem of development may change. These
patterns of development maJrbe attnbuted to endogenous FA ieveis, as 1s showi in Uiis stuciy, T h w e ~ u
c y
and down-regulated during cystocarp development.
Each stage is preceded by the transformation of existing
gametophytic celis into speciai protein-rich tissues, or
new secondary gametophytic tissues are formed that are
rich in protein contenL Fusion ceii formacion and organelle áutolysis is a process of senescence designed to
cannibalize compounds, partinilarly niaogen containing
substances, for use during the final stages of carposporc+
phyte development (Hommersand and Fredericq 1995).
lnis may expiain iower enciogenous ieveis of PAs
found in mature cystocarps of G. c m a . Endogenous
levels of PAs have not been related to physiological
events in algae, except for those descnbed by García-Timénez et d. (19981 and Marián et al. (2000a) on
morphogenesis during in uitro cuitivation of the red
algae Crateloupza ú q p h w a and Celidium cananenris.
It is also evident from the incubation experiment that
short exposure to P h in cystocarps at different develop
ment stages enhanced cell division and growth of carpospores, most probabiy due to the increased ceiiuiar
PAs levels. High levels of free PAs (PLT) have been reported in growing and dividing tissues of higher plants
(KaurSawhney et al. 1989). Moreover, when qstocarps
were incubated in PAs, they induced cystocarp maturation, seen as the num'ber of carposporangiai iines and
mature carpospores. particularly with SPM, in contrast
to rnatiiration process without PAs; as observed in the
histological sections. In the green algae Acetabulana
the inhibition of omithine descarboxylase, a key enzyme
of PA metabolism, profoundly affects the develop
ment of nucleate and anucleate fragments (Brachet et
al. 1978). Resides replation of +tocarp maturation
by endogenous PAs levels, our results also suggest that
PAs may promote carpospore release and further
growth and development. ln conclusion, PA levels during development of the
reprodvctive stnirt,ires ( c j t o c a r p ) in the red algae
G. conea increased from nonfertilized tissue (female
-.
ship of CONACyT (no. 112873). The fellowship of the Spanish
Plan de Formación del Personal Investigador u> Fernando Marián
is ako acknowledged.
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