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Effects of Oxygen Stress on ^1H-NMR RelaxationTime (T_1) of Vigna radiata Seedling

Iwaya-Inoue, MariLaboratory of Horticultural Science, Department of Plant Resources, Division of Bioresourceand Bioenvironmental Sciences, Graduate School, Kyushu University

Yoshimura, KazuhisaAnalytical Chemistry Laboratories, Department of Chemistry and Physics on Condensed Matter,Graduate School of Sciences, Kyushu University

Otsubo, MayukoLaboratory of Horticultural Science, Department of Plant Resources, Division of Bioresourceand Bioenvironmental Sciences, Graduate School, Kyushu University

Yamasaki, HideoLaboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus

https://doi.org/10.5109/24325

出版情報：九州大学大学院農学研究院紀要. 44 (3/4), pp.249-256, 2000-02. 九州大学農学部バージョン：権利関係：

,J. Pac. Agr., Kyushu Lilliv., 44 (:3'4) , 249-2:3(; (2000)

Effects of Oxygen Stress on 'H-NMR Relaxation Time (T,) of Vigna radiata Seedling

Marl lwaya-Inoue' *, Kazuhisa Yoshimura2, Mayuko Otsubo1

and Hideo Yamasaki 3

1 Laboralory of I lnrticultural ScieJl( :p' , I)rpartmcnt of Plant Resources, Divisi(.lII of Biorcsource and Biocnviromnent.al Sci~nccs, Graduate Srhool, Kyusltu Unive rsity,

RoppOllinatsu, t"ukuoka, 810-H:")GO JAPAN

~ Analytical Chelllistry Laboratories, Department. of Chemistry i'lnd Physics on Condensed Mat.ter, Graduate School of Sciences, Kyushu 1 :niversity,

Ropponmarsu , rukuoka, 810- RfifiO .JAPAN

~ LalJoratory of Cell and FUl lI:ljonal Hioiogy, Faculty of Science, University of t.he Ryukyus, Okinawa, 903-02U .JAPA ... 'J

(Recei'l)ed October 8, 1.9,1.).9 Ctnd accepted l'/O"l.'em.ber 5, 1999)

Effects of the oxygen exposure on plallts wer£> investigated ill inl ncr hypocotyllissu e::: llf etiolaLed seedlings from n llutg bean (Vigna md1jl1,o L.) . Vle report here 111M spin-lattice NMR

relaxation time (T,) of wa te,. proton of inl.a<.:t ti SSllf!S, which (Am lJe measur0d 'kith a 180 ' - r ~ 90

Q

Dulse spqllence at 20 MHz, is responsible to t ile oxy~en exposure. Wllell t.he tissues placed in t.hf' N~R tube wt: re exposed to 95% oxygen for 55, a rap id de(;n~asf' in the T , values a nd a subscquf!nl. rpj axation process were observed. The vallie was fully recovered t.o HIe initjal one dlUing a 40mill int uba tion. Heat-denat urt~d tissues did nor. show s uch r ecovery response, suggesting that the Tl recovery requires il ear,-sf!l1sitive cOTIlf)onenr.::; contained in the tissues. To examine a possibility that scaVPllging activit.ies of oxygen radicals is lllv()lw d in the mechanism [ur t.he 1', response, e rrect.s of supplemental scavengers ,vere t esreci. \Ve Iw.vc found that. the shortening of T, i.llduced by oxygen exposure to membrane fractions was st.rongly suppressed by lhe radical scavenger s upcraxide (iismu t<lsc (SOD) , Tiroll and ascorbate. These results s uggest thai activi t.if!s of O~· prod ucrion in (he !.issues are involved in lhe mechanism for abrupt shorlening of T ..

Key \'wrds: free raciical ~ mung he:tn ( Vi,yna radiata) - :-.J:-"1R re laxation lime (T,) -paramagnetic substance - slIpcroxide

Abbreviations: USA, bovine serum albumin; NMR, nuclear magneti c resonance; SOD, superoxide ciismllt.ase; 7 1, spin-lctt.tice relaxation time: T~ . spin-spin relaxati on time; Tiran, 4,5-dihydroxy- 1 ,1- Lenzene disulfnnic a c:iO.

INTR ODUCTION

Relaxation time (T1 or Tz) of water proton magnetic resonance in biological systems prmides important. clinical infonnation by virtue of the fact that relaxation mechanism against various stresses depends OlL the intrinsic state of water in cells (Kaku and fwaya-Inoue, 1990 refe rellces therein). Despite plants require molecular oxygen for

"'Correspond ing aillhor Mati Iwaya-Inoue. ma ri rcb@mbox.nc.ky\ lsh IHl.acjp

249

250 M. hoaya-Inuue el al

survival, oxygen can become a causative factor for stress and is toxic to plants, because it can be readily reduced to reactive oxygen species such as super oxide radicals (O~), hydrogen peroxide (H,O,) and hydroxyl radicals (OH) that can oxidize and damage cellular components (Halliwell, 1982; Hendry and Crawford, 1994). Oxygen radicals can react very rapidly v.ith DNA, causing severe cellular damages (Pinhero et al.) 1997). 0 toxicity to bacteria is also in part due to the activities of O2 formation in the tissues (Gregory and Fridovich, 1973b).

Spin~lattice relaxation time eTa is strongly influenced by paramagnetic substances, such as paramagnetic ions (Stout. el al., 1977; Ratko,,'c, 1987; Iwaya-Inoue et al., 1993), molecular oxygen (Lanir and Gilboa, 1981) and stable free radicals (Brasch, 1983) which possess large magnetic moments due to unpaired electrons in their orbital; and thereby exert a relaxing effect on neighboring hydrogen nuclei. Despite many studies on superaxide (VanToai and BoUes, 1991 references therein) '~ihich are also paramagnetic, they contain no information regarding the relationship between the production of superoxide and the water proton relaxation time. The present paper ¥.-ill discuss that the Tl can reflcct activities of O~- fannation in the plant. t.issues.

'vIATERIALS AND METHODS

Plant material Seeds of mung bean (Vigna mdiata (L.) Wilczek) kindly provided by Taishin

Jit.sugyo Co. Ltd., \-vere used as materials. Seeds were irmnersed into sodium hypochlo­ride (200 ppm) for one hour and were successively immersed in 1 mM CaSo. j solution at 25°C in the dark for about 15 h. Just. after the seeds initiated germination, they were seeded in cotton gauze unfolded on vinyllA-rire placed in a plastic pot (10 cm in diameter, 7.5 cm in dept.h). These pots were placed into a plastic vat. filled \vith 1 mM CaS04 solution at. 25"C in the dark. H.,.rpocotyls were excised on the 4 th day after germination and used for the experiments.

Measurements of proton TJ relaxation times The T j values of mung bean seeds \vere measured using a 180

0

- r _900

pulse sequence (Farrar and Becker, 1971) at 20MHz wit.h a Bruker Minispcc PC 20pulsed NMR spectrometer as described previously (Kaku and Iwaya-Inoue, 1988; Iwaya-Inoue et al., lDS9; 1993). The hypocotyl (about 4 ern in length) ofthrcc intact seedlings of mung bean \vas cut into t.VI/O pieces and then six pieces ViTere packed into a 7.5 rum diameter NMR tube. The probe temperature (20°C) was controlled by a thermost.at (Lauda Kryo-SK65) connected to the sample chamber of the spectrometer.

Oxygen exposure Effects of the oxygen exposure on T) for intact hypocoryl tissues of etiolated

seedlings from Intmg bean were compared between tissues denatured at 120"(; for 15m..in and control. Furthermore, the hypocotyl segments \vere homogenized at. O°C in a 30mM Hepes-tris solution (PH 7.5). V/hen the tissues in the NI\.-1R tube were exposed t.o 95% oxygen or nitrogen for 5 S, T) values for both control and heat-denatured tissues were immediately determined. In the case of fluid samples, ox~ygen or nitrogen was added to

O:rygen Stress and NAIR Relaxat~ion Thne 251

the fluids by bubbling gas in an NMR tube for 5 s, and T values for respective samples were also immediately determined.

Effects on TJ of addition of radical scavengers Centrifuged at IO,OO()Xg for 15min at 4 'C, the homogenate prepared in the same

way described above was separated into soluble and insoluble fractions. The soluble fraction obtained was filtered through a cellulose-acetatt-; filter (0.45,u.m, Advantec DISMIC-25). Plior to the exposure of oxygen, a mixture solution of 0.2 mglml SOD, 1 mM sodium ascorbate and 1 m1'I Tiron was added to each soluble or insoluble fraction obtained from fresh control and heat-denatured tissues. A BSA solution at the same concentration was llsed as a control for SOD. Effects on T j of the addition of radical scavengers to the soluble fractions, insoluble fractions and non-biological solutions were determined by using the samples mentioned above.

RESULTS AND DISCUSSION

Effects of oxygen exposure on spin-lattice relaxation time (T,) for intact tissues

INTACT TISSUES fresh tissues

o

o

o 15 30 45 60 75

TIME, min Fig. 1. The changes of TJ for intact hYl)Ocotyl t.issues of etiolated Vigna

n:uUa/,(l seedlings affected by hcat denaturing treatment and oxygen exposure. Fresh tissues CO) were exposed to (fJ5% v/v) oxygen for G s at the point indicated by arrm'i. Tissues ,verc suffered from heat-denatured treatment. (arrow head) at 120"C for 15min (.6.) and then eXIX)sed to oxygen for 5s (arrow). The probe temperature during all measurements was 20"C.

252 M. !uxLya- Inolw et a t.

The effects of the oxygen exposure OIl spin-lattice relaxation time (T!) for intact hypocotyl tissues of etiolated sccdling~ from mung bean \\!e I'C compared between ti ssues denatured at 120 'C for 15 min and control (Fig. I) . The T, of the hypocotyl of in tact. seedlings of mung bean \vas measured using 180

0 -, _90

0 pulse sequence hy pulse NMR

spectrometer as described previously ( Kaku and Iwaya-Inouc, 1988; Iwaya- Inoue el al. , 1989; 1993). Aft.e r the denaturing heat treatment, 'PJ values for iIl l()et t.isslIPS rl ~cn~aserl

from 1. 1 s to O.8 s. A similar tendency has also beell observed in frog lens t.i~sues exposed t.o heat stress by microwave (Nevill p. At nJ. , 1974). Even 40 ·C exposure to mung bean also induced a time-dependent decrease in T l ; the decrease in TJ values during the denaturing treatment seemed to depend on increases in protein content, macromolecular rearrangements and conformational changes that affect water-pro tein in te ract ion (Iwaya-[noue et at. , 1993). When the tissues in the t-: MR tube were exposed to 95% oxygen for 1) S, TJ values for both control and heat-denatured tissues immediately decreased. The local magnetic fie ld of rnoleeular oxygen (present as :10!) Vrill participate in decreasing 1'1 of wat.er proton due to the paramagnetic property of unpaired electron in their orbital: the magnetic susceptibility of oxygen molecule is J .35 X 10-" m'lkg, while that. of hydrogen molecule is -0.2 X 10-" m'lkg (Reitz and Milford, 1960 references therein). The suosequent relaxation behaviors were entirely different; the TL values for fresh control gradually increased to the init ial values during 40 min, while those for t.issHes suffcring from denaturing treatment remained low during Lhe same period. This evidenee suggests that some substances in fresh tissue::; prot.ect against the t.oxidty caused by t.he exposure of oxygen, while not in heat-denatured tissues.

Effects of oxygen exposure on TJ for tissue homogenate Fig. 2 sho\\lS time courses of Tl for fresh tissue homogenate and heat- denatured

tissue homogenate caused by the addition of oxygen. Tl values for the homugenate of the hypocotyl were significantly smaller LilaH lhuse uf water and a buffer solution , \'lhen o~ .. ygen was dissolved to Ule fluids by bubbling oxygen in an NMR tube for 55, Tl values for each solution immediately decreased and remained at low levels for 40 min Wltil the dissolved oxygen was displaced by 95% nitrogen. As soon as the addition of nitrogen, T , values of the four so~utions were almost restored to the initial levels or slightly higher levels because nitrogen is diamagne tic (Reitz and Milford, 1960). Apparently t.he relaxation behaviors are quite different from intact hypocotyl tissues and homogenate even obtain ed [rom fresh t issues, which have different intactness in cellular compartmentalization (Figs. 1 and 2).

The increased concentration of O~ lead to increasf'!d rates of O2 - production in yeast cells (Gregory et al .• 1974), and 0 ,- iB a COmmon product of the biological reduction of oxygen in many vascular plants (Bridges and Salin, 198 1). The induction of SOD is a response to the 0 , exposure (Gregory and Fridovich, 1973a) and son catalizes the following reaction: 20,-+2H+~0, +H,0, (McCord and Fridovich , 1969). In transgenic maize (Zea mays L.) v,.ith the highest FeSOD activit.ies plants enhanced tolerance toward oxidative st.ress and increased gro"th rat.es (Van llreusegem et ai., 1999). Therefore, the abrupt decrease in T, caused by the addit ion of oxygen probably depends either on the oxygen concentrat ion itself or the activities of O:!- production in fresh tissues, and hypocot.yl tissues are protected from toxic oxygen by the induction of SOD. Unlike the

TIME, min

Fig. 2. Effects of oxygen and nitrogen exposure on T, for a homogenate from mung bean hypocotyl tissues. Symbols are homogenate from fresh tissues CO), that from heat-denatured tissues (6), Jlon~bju­logical solutions sHch as water Ce) and a :30 mM Hepes-tris (pH 7.5) buffer solution C .... ). 95% oxygen was huhhlf'd int.o fluids for 5 s, subsequently niU'ogen for 5 s at the points indicated by arrows, respectively. The probe temperature during measurements was 20 "C.

paramagnetic inorganic substances such as MnH and Fe' 1 an unpaired electron in a free radical occupies the outermost molecular orbital and readily available for electron pairing wIth reducing agents (Brasch, 1983).

Effects all TJ of radical scavengers to the soluble fractions and insoluble fractions Negatively charged O2- carmot move across inside cells, but it is rapidly converted to

membrane-diffusible H,O, by SOD, ascorbate and thiols (Cheeseman and Slater, 1993). Effects on TJ of addition of radical scavengers to the soluble fractions, insoluble fractions and non-biological solutions were shown in Fig. 3. The addition of radical scavengers CO.2 mg/wl SOD, 1 mM sodium ascorbate and 1 mM Tiron) prior to the exposure of oxygen, was only effective to the membrane fractions obtained from fresh control tissues (Fig. 3B). BSA at the same concentration, which was used as a control for SOD, gave no pffect on the Tl for the insoluble fractions even prepared from fresh tissues. It has been \videly investigated that superoxide is produced in organelles such as chloroplasts CAsada, 1(184), mitochondria (Boveris, 1984) and glyoxisomes (Sandalio et at., 1988), while SOD

254

cf2. w~

:::I

~ 80 > ~ w > ~ m 60 a:

#. 100

W :::I ..J « > ~ w > ~ ..J W a:

80

60

M. Iwaya-Inoue et al.

SOLUBLE FRACTION A

control BSA SOD AA Tiron

INSOLUBLE FRACTION B

control BSA SOD AA Tiron

Fig. 3. Effects of radical scavengers on T, for soluble and insoluble fractions from fresh and heat-denatured tissues. Concentrations of BSA and SOD were 0.2 mg/ml, sodium ascorbate and Tiron were 1 mM, respectively. A: Soluhle fraction from fresh tissues (open hars), heat-denatured tissue (oblique bars) and buffer (closed bars). B: Insoluble fractions from fresh control (open bars) and heat-denatured tissue (oblique bars). Quoted values of Tl were expressed as relative Tl values of samples after the oxygen treatment to the initial ones. Tl values were the means of 7 to 10 replicates for each sample. The probe temperature during measure­ments was 20°C.

is in water-soluble fractions in plant cells (Giannopolitis and Ries, 1977). Since the water-soluble fractions in mung bean hypocotyl contain most of the matrix proteins (Iwaya-Inoue et ai., 1993), O2- was produced in the membrane fractions but not in the soluble fractions. TJ is not restored in heat-denatured tissues since the enzyme is known to be inhibited by 120°C treatment for 15 min and SOD is not induced (Nakano, 1988). These results support our idea that the TJ value is useful to monitor the O~ formation in intact tissues under stress conditions.

O:.rygen ,)'tress and NA-!R Relaxation Time 255

ACKN OWLE DGMENTS

We would like Lo express many thanks to Dr. Yoshinobu ivliyazaki of Fukuoka Universi ty of Education and Dr. Hideki Yayama of the Department of Chemistry and Physics on Conden~ed Matter, Graduate School of Sciences, at our university for useful discussion. \Ale also greatly appreciate Mr. Gary Mueller and Izumi Noda-Mucller for reading the English manuscriyt .

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