mechanism of pea leaf senescence (l), whereas mitochondria and chloroplasts may play an important role in the cellular toxicity mediated by activated oxygen ...
198s Biochemical Society Transactions ( 1 996) 24
Active oxygen metabolism in the senescence of pea leaves: ascorbate and glutathione contents in different cell compartments. A. JIMENEZ, J.A. HERNhDEZ, G.M. PASTORP, L.A. DEL RIO* and F. SEVILLA
---
-Fraction
Senescence days
Extract
Depto. de Fisiologia Vegetal, CEBAS, CSIC, Apdo 195, E30080 Murcia, Spain; *Depto. de Bioquimica, Biologia Celular y Molecular de Plantas. EEZ, CSIC, Apdo 419, E18080 Granada, Spain. Senescence is an oxidative process that involves, among other changes, the overproduction of active oxygen species. Recently, it has been proposed that peroxisomes can play a role mediated by activated oxygen species in the oxidative mechanism of pea leaf senescence (l), whereas mitochondria and chloroplasts may play an important role in the cellular toxicity mediated by activated oxygen species of NaCl in pea leaves (2,3). In this work, the presence of ascorbate peroxidase (APx) and the contents of ascorbate and glutathone in mitochondria and peroxisomes of dark-induced senescent pea leaves were studied. Pea (Pisum sativum L., cv Lincoln) leaves of 15-days- old plants were induced to senesce as described in (1). Mitochondria and peroxisomes were purified from young and senescent pea leaves as described in (4). The purified mitochondria and peroxisomes had intactness percentages between 70 and 85% and were free of chloroplasts as verified by the marker enzyme glucose-6-P dehydrogenase and the chlorophyll contents (data not shown). The APx activity was determined according to Hossain and Asada (5). Reduced Ascorbate (ASC), Glutathione (GSH) and oxidised forms DHA and GSSG, were detected by HPLC, according to (6,7). Although the presence of APx in chloroplasts and cytosol of pea leaves is well established (8), we have found A h activity in purified mitochondria (148 nmol asc. min-' mg-I) and peroxisomes (35 nmol asc. m s ' mg-'). The mitochondria1APx activity was 76% inhibited by 0.5 mM pchloromercuriphenil sulfonic acid. The mitochondrial and peroxisomal APx activity could represent the additional and tninor APx isozymes described in pea leaves by Mittler and Zilinskas (8). At day 11 of senescence, an increase in the level of MDA, taken as an index of lipid peroxidation, in crude extracts, mitochondria and peroxisomes from pea leaves, was observed (Table 1). Table 1 Lmd DeroxxbtIon l e d s
- s
-EL& value
rep-
-Fraction Emact Wtochondna Peroxlsomes
III
subceUuh fract~auof
the mean ofthree & &rat W MDA
senescence Qys 0
11 0 II
pmol x d-' 38 2 52 2 33 72
0
40
11
16 0
PRDA was &ermined as described in (9)
~
_
_
Table 2. Antioxidant contarts in subcellular fractions of senescent pea leaves. Pa& value represents the mean of at least three LfFerent experiments.
Mitochondria F'eroxisonies
-
ASC
0
7.10
I1 0 II
1.00
0 I1
DHA
wg mg protein
3.81 0.02 0.65 0.86
1 70 0.09 0.41
0.08 0 94
1.26
GSH
GSSG
nmol mg.' protein 9.70 0.02 0.61 0.93 5.92 0.21 3.52 0.22 4.15 0.15 33.60 51.77
At day 11 (Table 2) a strong decrease of total ascorbate content in crude extracts and mitochondria was observed, whereas in peroxisomes ascorbate was slightly increased and the ratio of DHNASC was maintained constant. In contrast, this ratio was increased in senescent pea mitochondria. In these organelles ghtatluone, like ascorbate, showed a decrease, of up to 40%, with senescence, and the ratio of GSSG/GSH was increased. However in peroxisomes, total @tathione content increased dramatically. This fact was rnainly due to the rise in the oxidised form, which was reflected in an important increase in the ratio GSSG/GSH, although the reduced glutathione pool was also enhanced. An increased production of 02.* radicals and Hz02 in pea leaf mitochondria during leaf senescence has been found (data not shown). These results and those described in this work, support the idea that some important oxidative injuries rnay occur in mitochondria during leaf senescence. The capacity of peroxisomes to maintain the ASC pools at c:ontrolled concentrations and to rise the pools of glutathione (GSH + GSSG), could help prevent oxidative injury in peroxisomes during leaf senescence. This is the first report on the presence of APx in leaf mitochondna and peroxisomes. These two cell organelles add to chloroplasts and cytosol where the occurrence of this antioxidant enzyme is well known (5, 8, 10) &knowledgements The authors are grateful to Mrs. D. Lapaz for technical assistance. "hJs work was supported by grants PB 92-0492C02 from the DGICYT (Spain) and CHRX-CT94-0605 from the E.U. FEFERENCES 1.- Pastori, G. M. & del Rio, L.A. (1994) Planta 193, 385-391. 2.-Hemhdez, J.A., Corpas, F.J., Mmez, M.; del Rio, L.A. & Sevilla, F. (1993). Physiol. Plant 89,103-1 10. 3.- H d d e z , J.A., Olmos, E.; Corpas, F.J., Sevilla, F. & del Rio, L..A.( 1 9 9 5 ) P h t SCi. 105, 151-167. 4. Struglics, A,, Fredlund, K.M., Rasmusson,A.G.& Moller, I . M. (1993) Physiol. Plant. 88, 19-28. 5. Hossain, M. A. & Asada, K. (1984) Plant & Cell Physiol. 25(7),1285-3295. 6.- Castillo, F. J. & Greppin, H. (1988) Env. and Exp. Botany 28, 23 1-238. 7.- Farber, Ch. M., Kanengiser, S., Stahl, R., Liebes, L & Silber, R. (1983) Anal. Biochem 134,355-360. 8.- Mittler, R & Zilinskas, B (1993) Anal Biochem 212,540-546 9.- Buege, J.A. & Aust, S.D. (1978) Methods Enzymol. 52. 302310. 10.- Mittler, R & Zilinskas, B (1994) Plant J . 5,397-405.
