Role of superoxide, lipid peroxidation and superoxide dismutase in ...

3 downloads 0 Views 2MB Size Report
Recalcitrant seeds of Shorea robusta (sal) exhibit 100"o viability up to 4 d after maturity. ... Key words: V'iability, Shorea robusta (sal), lipid peroxidation, superoxide radical, superoxide ..... the oil technologists association of India 13: 120.
Nezv Phytol. (1994), 126, 623-627

Role of superoxide, lipid peroxidation and superoxide dismutase in membrane perturbation during loss of viability in seeds of Shorea robusta Gaertn.f. BY K. S. KRISHNA CHAITANYA AND SUBHASH CHANDRA NAITHANI* School of Studies in Life Sciences, Pt. Ravishankar Shukla University, Raipur - 492 010 (M.P.), India {Received 9 March 1993; accepted 24 November 1993) SUMMARY Recalcitrant seeds of Shorea robusta (sal) exhibit 100"o viability up to 4 d after maturity. The rapid loss of viability after 4 d is associated with the reduction in moisture content below the lowest safe moisture content (37%). Seed becomes non-viable on 8 d. Increased leakage of electrolytes in seeds and lipid peroxidation in embryonic axes was discernible immediately from 0 d. In embryonic axes, very low levels of superoxide ('O^') were maintained up to 4 d and a sharp increase was registered up to 7 d. It is suggested that loss of moisture content in sal seeds below 21°.(, (after 4 d) induces substantial leakage loss probably due to increased lipid peroxidation and 'O.f radical formation which are responsible for severe membrane perturbations leading to rapid loss of viability. In embryonic axes SOD activity was recorded only in IOCVQ viable seeds. Key words: V'iability, Shorea robusta (sal), lipid peroxidation, superoxide radical, superoxide dismutase.

INTRODUCTION

Recalcitrant seeds are characteristically shed from the mother plant at higb moisture contents and, being desiccation-sensitive, lose viability at relatively high moisture contents. Investigations carried out on ageing sal {Shorea robusta Gaertn.f.) seeds (Purohit, Sharma & Thapliyal, 1982; Nautiyal & Purohit, 1985 a, 6; Tompsett, 1985) have revealed that these seeds undergo a high rate of desiccation (from 47 % to 6 ".Q)-Tompsett (1992) has shown the lowest safe moisture content (LSMC) for sal seeds was 30% to 40% and classified these seeds as recalcitrant. It has been suggested that the loss of 'structured water' in seeds could result in the disorganization of metabolism leading to the loss of stability of subceliuiar structures, including membranes leading to loss of viability (Farrant, Pammenter & Berjak, 1988). Loss of desiccation-tolerance and viability bas been studied at the ultrastructurai, biochemical and biophysical levels in several other seeds such as Acer platanoides (Hong & Ellis, 1990), Landolphia kirkii (Pammenter, Vertucci & Berjak, 1991), Zea mays • To whom correspondence should be addressed.

(Dev & Mukheriee. 1988; Leprince et aL, 1990). „, ' , , ,, , 1 he results suggest that cellular membranes are vulnerable to damage from desiccation and are probably the primary site of cellular injury (Sargent, Mandi & Osborne. 1981; Senaratna & McKersie, 1983). Seel, Hendry & Lee (1992) have shown oxidative damage (lipid peroxidation) in the desiccation-sensitive moss D. palustris when desiccated in the dark and concluded that the activated oxygen species are involved in desiccation damage. In saf, loss of membrane integrity and increased permeability during ageing is confirmed by enhanced leakage of electrolytes during imbibition (Nautiyal & Purohit. ]985f), yet the mechanism of membrane deterioration in sal seeds is not known. Besides mediating lipid peroxidation. 'O,"" and derived forms of activated oxygen are capable of oxidizing protein thiol groups, causing enzyme deactivation, initiating generation of more reactive and destructive species (Haiiiweil & Gutteridge, 1984), oxidizing nucleic acids (Pan & Yau, 1991). Tbe present study attempts to unravel the causes of rapid loss of viability by monitoring superoxide formation and dismutation and lipid peroxidation during loss of viability in sal seeds.

624

K. S. K. Chaitanya and S. C. Naithani

MATERIALS AND METHODS

Selection of seed Fully mature seeds (showing 100"o germination within 40-^8 h) oi" sal {Shorta robusta), 63 d after anthesis, were collected (Nautiyal Purohit, 1985 a, b) from Gariyabandh Forest (Raipur District) and brought to the laboratory within 4—5h; healthy seeds were stored in w'ell-aerated baskets under ambient conditions (40-45% R-H. and 27-32 °C). These seeds were analyzed for initial viability and moisture content and considered as '0 d' seeds. The biochemical analyses were carried out on the embryonic axes which are the location of major changes during loss of viability (Bewley, 1986).

(• O2 ) was measured by its capacity to reduce nitroblue tetrazolium (2 5 x 10"^ M). The absorbance of the end product was measured at 540 nm. 'O^^ formation was expressed as AA^^^/min/g f. wt of the sample. Superoxide dismutase activity

Weighed amount of embryonic axes were homogenized in ice cold borate buffer (sodium tetraborate-t-boric acid; 0-2 M; pH 74) containing 25 °o PVP (polyvinyl pyrollidone). The homogenate was centrifuged at 10000 rpm for 10 min. The supernatant was subjected to acetone precipitation at 0-4 °C (Naithani, 1987) and again centrifuged at 3000 rpm for 3 min; the pellet was resuspended in sodium phosphate buffer (0 02 M; pH 6 4) and used Germination as an enzyme source. Superoxide dismutase activity Seeds were surface-sterilized with HgCI^ (0-1 '\,), was determined by measuring the inhibition of thoroughly washed with distilled water (DW), pyrogallol autoxidation at 420 nm and quantified by allowed to imbibe DW for 24 h and germinated the method of Marklund & Marklund (1974). SOD on H^O-saturated filter paper in Petri dishes. activity was expressed as units of SOD tnin"^ g"' f. Germination was scored as radicle emergence to wt of the sample (embr>'onic axes). All spectrophotometric analyses were carried out using a 160-A 5-7 mm. UV-Visible Spectrophotometer (Shimadzu). Moisture content The moisture content of the seeds was determined using the formula given by International Seed Testing Association (1985):

RESULTS

Loss of viability

The ageing sal seeds show a rapid toss of viability, within 8 d of maturation (Fig. 1). Decline in fresh weight —dr\- weight percentage germination of the seeds was from 100"o xlOO. % moisture = fresh weight of the seed initially to 40"o on 6 d falling to 0"o by 8 d. A gradual decrease in the percentage moisture was also discernible. The n:\oisture content on 0 d was 49 "^ Specific conductivity and reduced gradually to about 30 "o on 6 d (Fig. 1). The leachates were collected after 24 h of imbibition Lowest safe moisture content (37—40%) for sal seeds of water by the seeds and the specific conductance (Tompsett, 1992) was recorded on 4 d (Fig. 1). After due to electrolytes loss by the seeds was recorded 6 d the moisture content decreased sharply and using a conductivity meter (Systronics). reached a minimum (6 %) at 10 d. Seeds from 0 d to 4d old showed 100% germination within 40-48 h and showed delayed germination with ageing after Lipid peroxidation 4d. Lipid peroxidation was measured as the concentration of thiobarbituric acid reactive substances, equated with malondialdehyde (MDA) (Heath & Packer, 1968) and expressed as AA54o/g f. wt of the sample (embryonic axes). The regression analysis for lipid peroxidation on % germination was obtained using the software, STATGRAPHICS, Version 2.1. Superoxide determination Weighed amount of embryonic axes were homogenized in cold (0-4 °C) sodium phosphate buffer (0 2 M, pH 7-2) containing diethyldithiocarbamate (10"^ M) to inhibit SOD activity. The homogenate was immediately centrifuged for 1 min at 3000 rpm. In the supernatant, superoxide anion

10

Figure 1. The decline in % germination and % moisture content with age of mature sal seeds. Eacb value is the mean of 50 observations. Inset: showing the positive correlation between loss of viability and desiccation (r = 0-924).

Membrane perturbation and viability of seeds of Shorea

0

2

4

625

6 Days

Figure 2. Loss of electToWtes from ageing sal seeds estimated by conductivity of the leachate. Values plotted are the mean of six replicates + SD.

Days

Figure 5- Changes in SOD activity in the embryonic axes of ageing sal seeds. Values are the mean of six replicates±SD. Maximum values of lipid peroxidation were recorded at 10 d (0-38 AA^^^ &"' f- '^'t) ^'ith the minimum of 014 AA^^^ g"'f. wt recorded on 0 d. The correlation between germination and lipid peroxidation is presented in Figure 3 (inset).

Superoxide formation 10 Days

Figure 3. Changes in lipid peroxidation activity in the embryonic axes of ageing sal seeds. Values are mean of six replicates + SD. Inset: showing the negative correlation between lipid peroxidation and loss of viability (r = —0-896).

The liberation of the superoxide radical ('Of) shows a low profile in the initial stages, but increases rapidly with the loss of viability, i.e. after 4 d. The minimum level of O^" was recorded at 2 d, and showed a gradual increase thereafter up to 4 d, after which the levels of "Og" rose sharply to the maximum at 7 d. Subsequently the liberation of the "Og" radical declines steeply in the embr>^onic axes (Fig. 4). SOD activity was low up to 1 d (Fig. 5) but then rose steeply up to 3 d. Thereafter, no SOD activity was observed on subsequent days.

DrscussiON

Days

Figure 4. Changes in amount of superoxide radicals in the embryonic axes of ageing sal seeds. Values are the mean of six replicates ±SD.

Electrolyte leakage The specific conductivity of the seed leachates increased with the loss of viability and a gradual increase was recorded from 0 d to 4 d with no further increase after 8 d (Fig. 2). Lipid peroxidation There was a consistent increase in the lipid peroxidation product (MDA) from 0 d to 8 d (Fig. 3).

The present study has confirmed the recalcitrant nature of sal seeds, rapid loss of viability being found when seeds desiccate below the lowest safe moisture content of 37"ci (Fig. 1). This agrees with findings of Tompsett (1992) who found a strong correlation between rapid loss of viability and lowest safe moisture content (30—^O'^o R.H.) in sal seeds. The increase in leakage loss of electrolytes, which is an indicator of increased membrane permeability (Simon, 1978; Bewley, 1986) was found during rapid loss of viability in sal seeds (Fig. 2) as also reported by Nautiyal & Purohit (1985.:). The role of lipid peroxidation in membrane damage and seed deterioration is well documented (Mead, 1976; Bewley, 1986; Wilson & McDonald, 1986). For desiccation - sensitive, recalcitrant or homoihydrous systems loss of viability and increased leakage loss have been linked to increased Upid peroxidation (Dhindsa & Matowe, 1985; Leprince et al., 1990; Seel et al..

626

K. S. K. Chaitanva and S. C. Naithani

in storage. In: Physiology of seed deterioration. CCSA Special Publication No. 11. Madison, USA: Crop Science Society of Annerica, 27—45. Dey G, Mukherjee RK. 1988. Deterioration of maize and mustard seeds: cbanges in pbospholipids and tocopherol content in relation to membrane leakiness and lipid peroxidation. Agrochimica Vat. XXXII, Nos 5-6: 430-439. Dhindsa RS. 1982. Inhibition of protein synthesis by products of lipid peroxidation. Fhytochemistry 21: 309-314. Dhindsa RS, Matowe W. 1985. Drought tolerance in two mosses: correlated witb enzymatic defenct- against lipid peroxidation. Journal of Experimental Botany 32: 79-91. Farrant JM, Pammenter NW, Berjak P. 1988. Recalcitrance a current assessment. Seed Science & Technology 16: 155-156. Halliwell B, Gutteridge JMC. 1984. Lipid peroxidation, oxygen radicals, cell damage and antioxidant tberapy. Lancet 23: 1396-1397. Harman GE, Mattick LR. 1976. Association of lipid oxidation witb seed aging and death, Nature 260". Ill-ZIA. Heath RL, Packer L. 1968. Pbotoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics 125: 189-198. Hong TD, Ellis RH. 1990. .\ comparison of maturation drying, germination and desiccation tolerarice between developing seeds of Acer psfudoplatanus L. and Acer platanoides L. New Phytologist 116: 589-596. International Seed Testing Association 1985. International rules for seed testing. Seed Science & Technology 13: 299-355. Leprince O, Deltour R, Thorpe PC, Atherton NM, Hendry GAF. 1990. The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize {Zea mays L.) New Phytologist 116: 573-580. Marklund S, Marklund G. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogaliol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 47: 469^71. Evidence of the generation of superoxide radicals Mead JF. 1976. Free radical mechanisms of lipid damage and conseqtiences for cellular membranes. In: Pyror WA, ed. Free due to desiccation has been documented (Seel et al., radicals in biology, vol. I. New York: Academic Press, 51-68. 1992). A similar desiccation-induced generation of Naithani SC. 1987. The roie of IAA oxidase, peroxidase and polyphenol oxidase in tbe fibre initiation on the cotton ovule. 'O.j" radicals is evident from our study, where a Beitrage zur Biologie der Pfiamen 62: 79-90. sharp increase in superoxide levels was found after Nasirullah T, Mallika, S, Rajalakshmi S, Vihhakar MN, 4 d, when moisture content was below the LSMC of Krishnamurthy, KV, Nagaraja, Kapur OP. \9^\. Journal of -370/ the oil technologists association of India 13: 120. In : Bringi N V, ed. Non-traditional oil-seeds and oils of India. 1987, India, 1-56. The free radical scavenging system, the enzyme Nautiyal AR, Purohit AN. 1985a. Seed viability in sal. I. SOD, showed activity for a short span, in the Pbysiological and biochemical aspects of seed development in embryonic axes, i.e. from 0 d to 3 d when 'O^" Shcjrea robusta. Seed Science &' Technology 13: 59-68, formation was low. It may be that the consequence Nautiyal AR, Purohit AN. 19856. Seed viability in sal, 11. Physiological and biocbemical aspects of aging in seeds of of the decline in SOD activity by 4 d is the rise in Shorea robusta. Seed Science & Technology 13: 69-76. *0™' after this time. N a u t i y a l AR, P u r o h i t A N . 1 9 8 5 c . Seed viability in sal. I I I . Membrane disruption in aging seeds of Shorea robusta. Seed Science & Technology 13: 77-82. Pammenter NW, Vertucci CW, Berjak P. 1991. HomeoACKNOWLEDGEMENTS hydrous (recalcitrant) seeds: dehydration, tbe state of water and viability characteristics in Landolphia kirkii. The authors wish to acknowledge thanks to Professor S. S. Plant Physiology 96: 1093-1098. Ali, Head, Department of Biosciences, Pt. Ravishankar Pan Shu-Mei, Yau Yuan-Yeu. 1991. The isozymes of superoxide dismutase in rice. Botanical Bulletin of Academia Shukla University, Raipur for providing the necessary Simca 32: 253-258. facilities. Financial assistance given to K.S.C. by the Purohit AN, Sharma MM, Thapliyal RC. 1982. Effect of Madhya Pradesh Council of Science & Technology, storage temperatures on tbe viability of sa! (Shorea robusta) and Bhopa! (B-47/90) is acknowledged. talura (Shorea talura) .seed. Forest Science 2^: 526-530. Rudrapal AB, Basu RN. 1982. Lipid peroxidation and membrane damage in deteriorating wbeat {Triticum aestivum cultivar Sonalika) and mustard (Brassica juncea cultivar B85) seeds. Indian Journal of Experimental Biology 20: 465—470. Sargent JA, Mandi SS, Osborne DJ. 1981. Tbe loss of REFERENCES desiccation-toierance during germination: an ultrastructural Berjak P, Pammenter NW, Vertucci C. 1992. Homoihydrous and biochemical approach. Protopla.ima 105: 225-239, (Recalcitrant) seeds; developmental status, desiccation sen- Seel WE, Hendry GAF, Lee JA. 1992. Effects of desiccation on sitivity and the state of water in axes of Landolphia kirkii Dyer some activated oxygen processing enzymes and antioxidants in Planta 186: 249 261. mosses. Journal of Experimental Botany 43: 1031-1037, Bewley JD. 1986. Membrane changes in seeds as related to Senaratna T, McKersie BD. 1983. Dehydration injury in germination and the perturbations resulting from deterioration germinating soybean. Plant Physiology 72: 620-624. 1992). In sal seeds, an increase in lipid peroxidation was recorded immediately after maturity, i.e. 0 d, reaching a maximum at 8 d and showing a significant inverse correlation with loss of viability (r = — 0-89) and desiccation (;• = —0-91) (Fig. 3). However, the stimulation of lipid peroxidation in 100°o viable (0 d to 4 d) embryonic axes may be attributed to peroxidation of the stored polyunsaturated fatty acids (Wilson & McDonald, 1986) which are present in large quantities in sal seeds (Nasirullah et al., 1981). Stored lipids have been reported in the embryonic axes of Landolphia kirkii (Berjak, Pammenter & Vertucci, 1992) whereas the dechne in fatty acid content due to peroxidation in the embryonic axes has been related to viability loss in pea seeds (Harman & Mattick, 1976). The extensive peroxidation of the large quantities of stored polyunsaturated fatty acids may itself contribute to loss of viability in sa] seeds, as the products of lipid peroxidation have been shown to have damaging effects in disorganization of metabohsm (Shimazaki et al., 1981; Dhindsa, 1982), leading to loss of viability (Rudrapal & Basu, 1982). Thus, we proposed that in sal seeds, the rapid loss of viability after 4 d may be due to the cumulative effect of the enormous peroxidation products of the stored polyunsaturated fatty acids and peroxidation of the mennbrane Hpids.

Memhrane perturbation and viability of seeds of Shorea Shimazaki K, Takeshi S, Kondo N, Sugahara K. 1981. Active oxygen participation in chlorophyll destruction and lipid peroxidation in sulfur dioxide fumigated leaves of spinach {Spinacia oleracea cultivar New Asia). Plant Cell Physiology 21: 1193-1204. Simon EW. 1978. Membranes in dry and imbibing seeds. In: Crowe JS, Clegg JS, eds. Dry biological systems. New York: Academic Press, 205-224.

627

Tompsett PB. 1985. The influenct of moisture content and storage temperature on the viability of Shorea almon, Shorea robusta, and Shorea roxburghii seed. Canadian Journal of Forest Research IS: 1074-1079, Tompsett PB. 1992. A review of tbe literature on storage of dipterocarp seeds. Seed Science & Technology 20: 251-267. Wilson DO jr, McDonald MB jr. 1986. The lipid peroxidation model of seed aging. Seed Science & Technology 14; 269-300.