Bailly, Christophe

Corbineau, Françoise

Côme, Daniel

Lehner, Arnaud

Rousseau, Sandra

English

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HAL Id: hal-02081252
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Changes in antioxidant enzymes during sunflower seed
development
Arnaud Lehner, Christophe Bailly, Sandra Rousseau, Françoise Corbineau,
Daniel Côme
To cite this version:
Arnaud Lehner, Christophe Bailly, Sandra Rousseau, Françoise Corbineau, Daniel Côme. Changes in
antioxidant enzymes during sunflower seed development. Journée de l’UFR de Biologie de l’Université
Pierre et Marie Curie, Paris 6, 2001, Paris, France. ￿hal-02081252￿
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CONCLUSII ON
The results obtained give new insights on acquisition of germinability and on changes in
antioxidant enzymes during sunflower seed development :
Desiccation tolerance is acquired very early in the seed developmental program (at least
when drying is mild);
Seed vigour increases after physiological maturity;
Although SOD and GR are active, they do not appear to be involved in acquisition of
desiccation tolerance or elaboration of seed vigour;
Catalase is a ssociated with seed desiccation through a transcriptional regulation of its
expression. CAT activity is correlated with the onset of physiological maturity but  probably not
with seed germinability.
CHANGES IN ANTIOXIDANT ENZYMES DURING SUNFLOWER SEED 
DEVELOPMENT
A. LEHNER, C. BAILLY, S. ROUSSEAU, F. CORBINEAU et D. CÔME
Physiologie Végétale Appliquée, Université Pierre et Marie Curie, Tour 53, 1er étage, 4, place Jussieu, 75252,Paris, cedex 05.
IINTRODUCTII ON
Seed germinability and vigour are closely related to the conditions of seed development and
maturation on the mother plant. The development of orthodox seeds is associated with
reserve accumulation, which provides the energy source for seedling growth, and with a
pronounced desiccation phase, which allows the seeds to enter a dry quiescent state.
Desiccation of plant tissues has been shown to be related to production of active oxygen
species (AOS), the accumulation of wh ich may generate various cellular damage (Leprince et
al, 1 993; Smirnoff, 1993). Therefore, the abili ty of seeds to escape oxidative injuries during
their programmed de siccation on the mother plant, through AOS scavenging, might be related
to their subsequent germinability. The aims of this work were (I) to study the changes in the
antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase
(GR) during sunflower seed development and after artificial drying, and (II) to i nvestigate
whether there was a relationship between acquisition of seed vigour and expression of these
enzymes.
MATERIIIAL   AND   METHODS
Plant materialÊ: Experiments were carried out with seeds of sunflower (Helianthus annuus L., cv Fructidor) issued from cut
capitula th at were hand collected 24 to 58 DAP (days after pollination) in 1999 in experimental fields of Limagrain (Dr™me,
France). Seeds were either frozen in liquid ni trogen immediately after threshold and stored at Ğ35¡C (fresh seeds) or dried
on the capitula at ambient temperature for 3 days and stored at 20¡C  and 75 % relative humidity (RH) (dry seeds).
Germination tests : Germination was tested according to International seed Testing Association (ISTA) rules by estimating
the percentage of normal seedlings. Mean time to germination (MTG) was determined at 15¡C  using the following formula :
MTG = [ n11 + (n2-n1)2 + ... .+ (n j-nj-1)nj] / r (where nj represents the number of germinated seeds at d ay j a fter sowing,
and r represents the total number of germinated seeds).
Controlled deterioration : Seeds, equi librated at a moisture content of 8 % ( fresh weight basis) by placing them at 20¡C  in
75 % RH for 7 days, were placed for 3 to 7 days at 45¡C. Subsequent germination was evaluated according to ISTA rules.
Enzyme extraction and assayÊ: Extraction of proteins and measurements of the activi ties of catalase (CAT, EC 1 .16.1.6)
and glutathione reductase (GR, EC 1.6.4.2) were carried out as previously described (Bailly et al., 1996). Results are
expressed as specific activity (per mg of protein).
RNA isolation and northern blot analysis : RNA extraction was carried out a ccording to the method described by Verwoerd
et al. (1989). Ten g of total RNA, fo r each sample, were loaded on gel, transferred on to blot and then hybridizated with a
partial cDNA of CATA1 gene (clone L28740 purchased by Pr Heinze, MŸnster University).
RESULTS
SEED DEVELOPMENT
Seed dry mass accumulated from 24 to 42 days after pollination (Fig. 1). At the end of reserve
accumulation, so called physiological maturity, thousand seed weight (TSW) was ca 55 g and
moisture content was aro und 30 % fresh weight (FW). Seed water content regularly
decreased from 60 % FW at 24 DAP to 10 % FW at 58 DAP, thus showing no marked
desiccation phase.
Table I : Germination ability and seed vigour of dried seeds collected at various stages of their development. MTG,
mean time to germinate. T50, duration of controlled deterioration which reduced seed germination by 50 %.
Figure 1Ê: changes in moisture content () and in thousand seed weight (TSW, ) o f sunflower seeds
during their development.
Days after pollination
ELABORATION OF SEED GERMINABILITY
Dry seeds were able to germinate, and t hen to tolerate desiccation, as soon as 24
DAP, ie when they were still  immature (Table I). Germination percentages increased
during seed development until seeds reached physiological maturity. Further
decrease of percentages of normal seedlings was related to fungus contamination.
Seed vigour, as assessed by germination rate at 15¡ C and tolerance to controlled
deterioration, improved during seed development and after physiological maturity
(Table I). Indeed, MTG decreased and duration of controlled deterioration necessary
to reduce seed germination by 50 % (T50) increased during development.
CHANGES IN ENZYME ACTIVITIES DURING SEED DEVELOPMENT
GR activity did not evolved significantly during seed development on the mother plant
and was very similar in fresh and dry seeds (Fig. 2A). SOD activity displayed the
same profile of activity and therefore did not show any significant change during seed
development (data not shown).
CAT activity increased markedly at t he end of s eed fill ing, ie af ter 42 DAP, and
remained thereafter at a high level, close to 10 nmole H2O2 m in
-1
 mg protein
-1
 (Fig.
2B). Drying of im mature seeds was associated with a stimulation of th is enzyme
activity, which reached a value close to that found in mature seeds. The effect of
dehydration on CAT activity was not observed after physiological maturity, when seed
moisture content was low.
DAP Normal
seedlings
(%)
MTG at
15¡C (h)
T50 (h)
24 89,7 80 122,4
34 97,7 64 132,0
42 95,3 57 144,0
50 90,7 47 156,0
58 79,3 47 170,4
Figure 2 : Changes in glutathione reductas e  (A) and catalase (B) activities in seeds collected at various stages  of
development. Measurements were carried out in fresh seeds () and in dried seeds  (). Means of 3 measurements.
Verticals bars denote standard deviations.
Days after polli nation
A B
CHANGES IN CATALASE EXPRESSION DURING SEED DEVELOPMENT
Expression of one of the four genes coding for CAT, CATA1, has been investigated in seeds
collected from capitula 29, 42 and 58 DAP (Fig. 3). In fresh seeds, CATA1 expression
increased continuously during desiccation, and reached values close to 80 arbitrary units (AU)
in fully  mature seeds (58 DAP). CATA1 expression was stongly stimulated by artificial drying
of seeds at 29 and 42 DAP, reaching values close to that found in fully  mature dry seeds (Fig.
3). Drying of seeds at 58 DAP did not change CATA1 expression.
REFERENCES
BAILLY C., BENAMAR A., CORBINEAU F., CïM E D. (1996) Physiol. Plant., 104, 646-652.
ISTA (1993) Seed Sci. Technol. 21, Supplement , 141-186.
LEPRINCE O., HENDRY G. A. F. and Mc KERSIE B. D. (1993) Seeds. Sci. Res., 3, 231-246.
SMIRNOFF N. (1993) New Phytol. 125, 27-58.
VERWOERD T. C., DEKKER BÊ. B. M. et HOEKMAN A. (1989) Nucl. AC. Res., 17, 2362.
Figure 3 : A, Northern hybridization of CATA1 mRNA prepared from fresh and dried seeds collected at
29, 42, 58 days after pollination (DAP). B, electrophoresis gel of the membrane shown in A. C,
Hybridization signal intensity data from northern hybridization of CATA1 gene in the fresh () and dried
() seeds during their development. collected at 29, 42 and 58 DAP.
Days after pollination
        Fresh seeds                  Dried seeds
DAP               29      42      58         29      42      58
A
B
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Changes in antioxidant enzymes during sunflower seed development

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Changes in antioxidant enzymes during sunflower seed development

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