adaptation strategies in wetland plants: links between ecology and physiology. proceedings of a...

The Effect of Post-Hypoxia on Roots in Senecio and Myosotis Species Related to theGlutathione SystemAuthor(s): Sophia Biemelt, Gerd Albrecht and Ernst-Manfred WiedenrothSource: Folia Geobotanica & Phytotaxonomica, Vol. 31, No. 1, Adaptation Strategies in WetlandPlants: Links between Ecology and Physiology. Proceedings of a Workshop (1996), pp. 65-72Published by: SpringerStable URL: http://www.jstor.org/stable/4181417 .

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Folia Geobot. Phytotax. 31: 65- 72, 1996

THE EFFECT OF POST-HYPOXIA ON ROOTS IN SENECIO AND MYOSOTIS SPECIES RELATED TO THE GLUTATHIONE SYSTEM

Sophia Biemelt, Gerd Albrecht & Ernst-Manfred Wiedenroth

Humboldt-Universitdt zu Berlin, Institut fiir Biologie, Botanik und Biologie-Didaktik, Spathstr. 80/81, D- 12437 Berlin, Germany; tel. +4930 6329941, fax +4930 6329446, +4930 6328306

Keywords: Glutathione, Glutathione reductase, Hypoxia, Post-hypoxia

Abstract: This paper shows the effect of re-aeration following hypoxic pretreatment on the glutathione system in plants with different flooding tolerance. Re-aeration of hypoxically pretreated roots led to an increase Of TBA-rm content indicating an accelerated lipid peroxidation (post-anoxic injury). Re-admission of oxygen resulted in a clear increase in the content of total glutathione in both flooding-intolerant species Myosotis arvensis and Senecio jacobaea. Simultaneously, the high ratio between reduced (GSH) and oxidized (GSSG) glutathione decreased in these species upon the onset of re-aeration, while the tolerant Myosotis palustris and Senecio aquaticus showed only little changes in contents of GSH and GSSG. An imbalance in GSHIGSSG ratio reflects oxidative stress. The glutathione reductase (GR) reacted very differently in the investigated genera. The metabolic response to varying oxygen pressure is much stronger in the flooding-intolerant species compared to species naturally growing in wetlands. The present results suggest that glutathione system is an important component in overcoming oxidative stress.

INTRODUCTION

Recently, it has been shown that re-exposure to oxygen after a period of oxygen deprivation can cause serious injury (CRAWFORD 1992, ALBRECHT & WIEDENROTH 1994). This phenomenon, called "post-hypoxic stress", arises particularly from the generation of reactive oxygen species. In order to estimate the ability of plants to adapt to flooding conditions we therefore have to consider both hypoxic and post-hypoxic events.

Although the formation of active oxygen species, such as singlet oxygen, superoxide and hydrogen peroxide, is generally considered to be detrimental to cellular function, these molecules are formed in normal cell metabolism as by-products of biological redox reactions. These radicals can initiate lipid peroxidation and attack macromolecules like proteins, carbohydrates and DNA. The toxicity of active oxygen is counteracted by a highly efficient antioxidative defence system composed of non-enzymatic (ascorbate, tocopherol, glutathione) and enzymatic (superoxide dismutase, catalase, GR) constituents present in all plant cells.

The normal aerobic life is characterised by a steady state of formation of reactive oxygen species and their consumption by antioxidants (SIES & MURPHY 1991). On the other hand, increased production of toxic oxygen species is considered to be a common feature of stress conditions (FOYER et al. 1994). An increase in the activity of antioxidative enzymes, and in the level of antioxidants such as ascorbate and glutathione, is interpreted to be advantageous to overcoming stress situations. In the present paper we want to estimate:



66 S. Biemelt et al.

(a) the degree of post-hypoxic injury in plant species with different flood tolerance, and (b) whether the glutathione system of roots is involved in overcoming oxidative stress. Although the glutathione system is well characterized for the light-dependent oxidative

stress in leaves (FOYER & HALLIWELL 1976, ASADA 1992), up to now there has been little detailed information about glutathione metabolism and the function of glutathione reductase (GR) in roots. Glutathione can react with singlet oxygen and hydroxyl radicals and protect the thiol groups of enzymes. The reduced form of glutathione (GSH) is mainly involved in the regeneration of ascorbate within the ascorbate/glutathione pathway. In such reactions, glutathione is oxidized to glutathione disulfide (GSSG). GSH is regenerated by glutathione reductase (GR) in a NADPH-dependent reaction. Fig. 1 reflects the interplay of glutathione and ascorbic acid. We measured the content of reduced and oxidized glutathione and the activity of GR under hypoxia and post-hypoxia in roots of species with different flooding tolerance. Running experiments show the involvement of ascorbic acid in the detoxification cycle in roots as firstly documented by DALTON et al. (1991). Myosotis palustris (L.) L. em. RCHB. and Senecio aquaticus HILL as plants from flooding prone sites have been compared with Myosotis arvensis (L.) HILL and Seneciojacobaea L. from habitats with only a low risk of oxygen shortage. Additionally, the content of TBA-reactive material, indicating the process of lipid peroxidation, was measured in order to show the connection between post-hypoxic injury and the generation of activated oxygen species.

Abbreviations: GSH - Glutathione, reduced form; GSSG - Glutathione, oxidized form; GR - Glutathione reductase; TBA-rm - 2-thiobarbituric acid reactive material.

MATERIALS AND METHODS

Plant growth

Plants were grown in a growth chamber with day/night-alteration (day: 16 h, 22 ?C, 2 1 280 ,umol m2 s- PAR; night: 8 h, 17 ?C) over the whole time. Seeds of Myosotis arvensis,

Myosotis palustris, Senecio jacobaea, Senecio aquaticus were germinated on moist filter paper and, after 14 days, were transfered to vermiculite-substrate. At the age of 6 weeks, seedlings were placed in Eppendorf-tubes in plastic-vessels containing Knop-nutrient solution flushed with air. After 7 days, some of these plants were further flushed with air (C-control, oxygen pressure >20 kPa). Another group of plants were flushed with pure nitrogen (HI - hypoxia-inducing conditions, no oxygen detectable in the nutrient solution). Following 12 days of these treatments the plants were used for the experiment. Intact plants of HI-variant were removed to a nutrient solution with 20 kPa oxygen pressure for different periods of re-aeration (20 min, 2h, 16 h).

Assay of glutathione

The 0.5 g fresh weight samples of the roots were homogenized with sand and 5 ml of 5% sulphosalicylic acid. Cellular debris and sand were removed by centrifugation at 15000 rpm for 15 min. The supernatant was used directly for assaying total glutathione. Oxidized glutathione (GSSG) was determined following removal of reduced glutathione. Then, 1.8 ml of the extract was incubated with 120 gl vinyl pyridine for 1 h at 23 ?C. The vinyl pyridine was removed by three extractions with ether. The resultant solution contained GSSG. The



The effect of post-hypoxia on roots 67

H202 ASC NADPH NAD- \NADP

DHA

Fig. 1. Pathway of ascorbate/glutathione-system (following DALTON et. al 1993). Abbrevations: ASC - ascorbate, MDHA - monodehydroascorbate, DHA - dehydroascorbate, GSSG - glutathione, oxidized form; GSH - glutathione.

standard incubation mixture and the experimental scheme followed SMITH et al. (1984). The change in absorbancy was recorded at 412 nm for 10 min.

Determination of glutathione reductase

Glutathione reductase activity was measured by the modified method of FOYER & HALLIWELL (1976). Roots (0.5 g fresh weight) were ground with pestle and mortar using sand and 5 ml of phosphate buffer (pH 7.1-7.3). The supernatant was recovered by centrifugation at 17000 g for 15 min. The reaction was initiated by the addition of 100 ,l of 0.2 mM NADPH to 200 g1 extract in a 2.1 ml Tris-HCl-buffer containing 100 g1 of 0.5 mM GSSG. The rate of oxidation of NADPH was measured at 340 nm for 10 min.

Assay of TBA-rm

TBA-rm (2-thiobarbituric acid-reactive material) in root tissue was assayed according to the modified method of HEATH & PACKER (1968), using 0.2 g fresh wt. in 5 ml 0.3% 2-thiobarbituric acid in 10% TCA. After heating at 95 ?C for 30 minutes the mixture was centrifuged at 2000 g for 15 minutes. The absorbancy of the supernatant was measured at 532 nm and corrected for non-specific absorbancy by subtracting the value at 600 nm (DE VOS et al. 1989).

RESULTS

Lower values of TBA-rm were measured for all species under hypoxia-inducing conditions compared to aerobically grown roots (Fig. 2). Re-exposure of roots to air led to an increase of TBA-rm content reflecting an accelerated lipid peroxidation. In the flooding-tolerant species Senecio aquaticus, the maximum was reached after 2 h of re-aeration, whereas it was not




a

U mWosodsust,iws _ 0.5 0. o-

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30 HI HI+20'C Hl+2hC Hl+16hC C

b Q Seneclojacbaea

Seneclo aquaticu

0.8 -

i.0.6-

~0.4-

2 0

.J HI HI+20'C HI+2hC HI+16hC C

Fig. 2. Level of lipid peroxidation measured as TBA-rm in roots of plants grown under hypoxia (HI) and following re-aeration for 20 min (HI+20'C), for 2 h (HI+2hC) and for 16 h (HI+l6hC) compared with aerated control (C). Each data point represents the mean (? s.d.) of at least five independent experiments. a - genus Myosotis; b - genus Senecio.

reached until 16 h in the other species. Lower levels of total glutathione have been shown for all species for hypoxically grown roots compared with aerobically grown controls (Tab. 1). The flooding-tolerant species Myosotis palustris and Senecio aquaticus possessed higher levels (compared with the flooding intolerant species) for both cultivation treatments.

Re-admission of oxygen resulted in a significant increase in the content of total glutathione in the very flooding-sensitive species Myosotis arvensis and also to a considerable extent in Senecio jacobaea. Simultaneously, the high ratio between GSH and GSSG decreased upon the onset of re-aeration of the ambient root medium in Myosotis arvensis. This is mainly attributed to the slightly higher levels in the content of GSSG (Tab. 1). In comparison, Myosotis palustris and Senecio aquaticus showed only small changes in contents of GSH and GSSG in response to re-aeration. Concerning the activity of glutathione reductase (GR), the investigated genera reacted very differently. Hypoxically pretreated roots of the




Table 1. Effect of the re-aeration time (20 min, 2 h, 16 h) on glutathione content [nmol/g fresh wt.] in the root systems of the plant species precultivated in nitrogen-flushed (HI) nutrient solution compared with the continuously aerated control (C) and plants grown in hypoxia only (HI). Standard deviations are in parentheses (n=5): GSSG - glutathione, oxidized form; GSH - glutathione.

HI HI+20'C HI+2hC HI+l6hC C

Myosotis arvensis GSH +GSSG 52(25) 112(41) 100(47) 52(40) 65(27) GSSG 1(1.8) 7.2(4.1) 5.8(5.0) 3.5(3.5) 4.8(3.3) GSH 51 105 94 48 60 GSH/GSSG 51 15 16 13 12

Myosotis palustris GSH+GSSG 60(17) 67(21) 54(13) 45(22) 81(41) GSSG 3.3(3.2) 4.3(4.0) 3.2(2.9) 3.5(3.0) 4.7(2.7) GSH 57 63 51 41 76 GSH/GSSG 17 15 16 12 16

Senecio jacobaea GSH+GSSG 131(35) 163(47) 165(42) 174(55) 201(39) GSSG 4.5(3.6) 6.4(5.4) 5.5(3.7) 6.1(4.2) 9.4(6.7) GSH 126 156 159 168 192 GSH/GSSG 28 24 29 28 20

Senecio aquaticus GSH +GSSG 183(63) 189(78) 189(62) 206(74) 249(54) GSSG 12.8(12.5) 10.8(10.6) 7.2(5.4) 9.5(8.1) 14.4(13.9) GSH 170 178 182 196 235 GSH/GSSG 13 16 25 21 16

Senecio-species contained a lower enzyme activity in comparison with the aerated controls. The activity of GR increased immediately after re-exposure to aerated nutrient solution. The flooding-sensitive Senecio jacobaea responded with an increase in the activity of GR of approximately 65% while in S. aquaticus the increase was only 20% (Fig. 3b). In species of the genus Myosotis, the activity of this enzyme was greater under deprivation of oxygen compared to the aerated controls (Fig. 3a). This difference was particularly remarkable in the flooding-intolerant Myosotis arvensis. Hypoxically grown roots of M. palustris showed only a 4-fold higher enzyme activity than aerobically grown ones reaching the values of the aerated control after 20 minutes exposure to air. In contrast the GR activity of M. arvensis was 20 times higher in hypoxic compared to aerobically grown roots and recovered only slowly after the onset of re-aeration never reaching the values of the control during the investigated period (Fig. 3a).

DISCUSSION

Little is known about the response of protecting mechanisms in roots which were deprived of oxygen and subsequently re-aerated. The capacity to minimize post-anoxic injury appears to lie in the ability of plants to use a range of defence mechanisms such as antioxidants or




a

2.0I 1.5 - 1.0 0.51 li-I

0 0.251- * A43o0s arn818 0.2 - K Pvsofl spallf

0.15 - __\ _0.1 - \

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0 1 2 16

trie of re-aeraton [h]

b

0.25-

C

* 0.2

(!~ 0.15

0.05 * Senediojacobaea E U Senedo aquatius

0- I 0 1 2 16

time of re-aeration [h]

Fig. 3. Effect of re-aeration of the ambient root medium on glutathione reductase (GR) activity [gmol/g fresh wt.] in plants grown in nitrogen-flushed nutrient solution (closed symbols) compared with aerated control (open symbols). Each data point represents the mean (? s.d.) of at least five independent experiments. a - genus Myosotis; b - genus Senecio.

radical scavening enzymes including the ascorbate/glutathione-cycle to eliminate potentially harmful active radicals. The assumption that stress tolerance may be improved by increasing the endogenous antioxidant capacity of plants was considered under two aspects:

(1) as the occurrence of oxidative damage to membranes and other cellular constituents measured as TBA-rm;

(2) as the enzyme activity of the antioxidative system and in the levels of glutathione in response to hypoxia and re-aeration.

The increasing content of TBA-rm in all four investigated species following re-aeration provides evidence for imposed injury affecting the unsaturated fatty acids of the membranes.




The process of lipid peroxidation apparently seems to be suppressed when root tissues are deprived of oxygen probably due to the "shortage" of activated oxygen species.

The level of glutathione was lower under hypoxia inducing conditions in all tested species. Our observation that the increase in total glutathione after the re-admission of oxygen was more pronounced in the flooding sensitive species Myosotis arvensis and Senecio jacobaea is in line with the results of SCHMIDT & KUNERT (1986) and ALBRECHT & WIEDENROTH (1994), describing a sharp increase in glutathione as a gauge for stress situations in plant tissues. The most sensitive of the four tested species Myosotis arvensis doubled the level of total glutathione within 20 minutes, while Myosotis palustris showed no differences during the 1 6h re-aeration period. The increase in glutathione seems to reflect the hazardous return to an aerobic environment. Re-aeration leads to a slight decline of the ratio between reduced and oxidized form of glutathione in both flooding-intolerant species, which is a further sign for oxidative stress.

In contrast to the results on wheat seedlings (ALBRECHT & WIEDENROTH 1994), in the genera Senecio, and especially Myosotis, the glutathione reductase seems not to be the limiting step in reduction of the glutathione pool. We have always found a high reduction level of glutathione, only slightly changed immediately following the re-exposure to oxygen. A high reduction stage of the glutathione pool is known to be necessary in achieving optimal protein synthesis (RENNENBERG 1982). The reported high GSH/GSSG ratio for roots even after 12 days of oxygen shortage provides further evidence for metabolic adaptations of the roots to maintaining the cellular reduction state. These values are consistent with the assumption that more than 95% of glutathione is usually present in the reduced form (BIELAWSKI & JOY 1986, RENNENBERG & BRUNOLD 1994). The necessarily high reduction stage of glutathione in the sensitive species M. arvensis seems to be achieved at the cost of a remarkably high GR-activity compared to flooding-tolerant M. palustris. In M. arvensis this enzyme is forced to work at high activity under hypoxia inducing conditions in order to keep the balance between the GSSG and GSH. The 20-fold higher activity of GR in M. arvensis, under oxygen deprivation, compared with the aerated control is interpreted as a sign of great environmental strain. In comparison, GR-activity increased only 4 times in the flooding tolerant M. palustris under oxygen shortage. Among the Senecio species, the intolerant S. jacobaea reacted more sensitively than the tolerant S. aquaticus. The higher enzyme acivity is necessary for keeping the balance in the GSH/GSSG ratio. So the response of GR to hypoxia and subsequent re-aeration is a very individual one.

The results indicate that metabolic changes in response to oxidative stress are more pronounced in the flooding-intolerant species compared with the related flooding-tolerant ones which can also maintain their metabolic equilibrium under such stress conditions. Depending on the life strategy and habitat of the species, many different mechanisms can be involved to ensure the survival of the plants under varying oxygen pressure. The glutathione system is one of these mechanisms counteracting the rapid peroxidative damage improving the plant tolerance against changes in oxygen availability. Although the present studies have not yet provided sufficient information on the capacity of roots to cope with the harmful effects of oxygen radical production we conclude that, among the different levels in protection systems against oxygen radicals, the glutathione system is one of the fundamental markers for oxidative stress in plant roots.




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