ORIGINAL ARTICLE

In vitro evaluation of mitochondrial–chloroplast subcellularlocalization of heme oxygenase1 (HO1) in Glycine max

Shubham Dixit & Khushbu Verma & Gyan Singh Shekhawat

Received: 3 August 2013 /Accepted: 10 October 2013# Springer-Verlag Wien 2013

Abstract Heme oxygenase1 (HO1) catalyzes the degradationof heme in to biliverdin, carbon monoxide, and ferrous ions. Itsrole in higher plants has been found as an antioxidant andprecursor of phytochrome synthesis. The present study focuseson subcellular localization of HO1 in leaves of soybean hasbeen investigated. Most activity appeared to be located withinchloroplast due to its role in phytochrome synthesis butmitochondria also share its localization. Mitochondrial locationof HO1 might be on its inner membranous space due to its rolein the synthesis of electron donor species which facilitates HO1catalyzed reaction. Study reports the co-localization of HO1 inboth chloroplast and mitochondria.

Keywords Heme oxygenase1 . Subcellular . Localization .

Mitochondria . Chloroplast .Glycinemax

Introduction

Heme oxygenase (HO, EC 1.14.99.3) is a regulatory enzymethat cleaves heme to biliverdin IXa (BV), with the concomitantrelease of carbon monoxide (CO) and the production of freeiron (Fe2+). Afterward, BV is reduced to bilirubin (BR) throughthe action of BV reductase. Both BV and BR are potentantioxidants (Llesuy and Tomaro 1994). Further resultssuggested that HO consists of two types of isoforms as follows:an inducible heme oxygenase1 (HO1) and a constitutive hemeoxygenase-2/3 (Maines 1988). Until now, genes encoding

HO1 have been isolated and characterized in many organismsincluding humans, animals, red algae, cryptophytes,cyanobacteria, and pathogenic bacteria (Shekhawat and Verma2010; 2011). These HO1s perform a wide variety of cellularfunctions and exhibit different enzymatic characteristics. Inmammals, for example, HO1 and its catalytic products areimportant in neural function and in protection against oxidativestress and tissue injury (Tomaro and Batlle 2002). HO1 is alsorequired for the synthesis of the phycobilin chromophores incyanobacteria and algae and plants (Terry et al. 2002).Similarly, recent studies in plant have found that HO1 isresponsible for phytochrome synthesis (Davis et al. 2001). Itcould also be induced by multiple stimuli and various abioticstresses, such as heavy metals, glutathione depletion, UVradiation, salinity stress, and hydrogen peroxide (Hanet al. 2008; Wu et al. 2011; Cao et al. 2011, Shen et al.2011, Li et al. 2012; Yannarelli et al. 2006; Balestrasse et al.2008, Chen et al. 2009, Bose et al. 2013). These responseswere thought to be a part of cellular defense mechanismagainst various stress-triggered oxidative damages and alsoexhibit hormone-like responses.

The subcellular localization of heme oxygenase1 in plant cellhas been an important part of discussion after finding its presentin plant system. Since in the past, light has been implicatedhaving amajor role in phytochrome synthesis, it appeared logicalto assume that heme oxygenase is localized in chloroplast whichis later on confirmed as well (Watanabe et al. 2001). But hemeoxygenase1, which is a metalloprotein, catalyzes the rate-limiting step in oxidative degradation of heme (Shekhawat andVerma 2010) also confers protection against abiotic stress. Themechanism bywhichHO1 provides protection likely involves itsenzymatic reaction products: CO, biliverdin IX-α, and ferrousion. The transcriptional induction of HO1 responds to a widevariety of cellular oxidative stress. Oxidative stress leads to theburst of reactive oxygen species (ROS), which is an inducer ofheme oxygenase1. A significant portion of cellular ROS arises inmitochondrion, associated with the leakage of partially reduced

Handling Editor: Bhumi Nath Tripathi

S. Dixit :K. VermaDepartment of Bioscience and Biotechnology, Banasthali University,Banasthali 304022, India

G. S. Shekhawat (*)Department of Botany, Jai Narain Vyas University,Jodhpur 342001, Rajasthan, Indiae-mail: gyans.shekhawat@gmail.com

ProtoplasmaDOI 10.1007/s00709-013-0569-9

oxygen from the ETS under normal and stressed condition. Sothere might be strong possibility of HO1 localization inmitochondrion as well. Furthermore, heme degradation reactioncatalyzed by HO1 is an energy-dependent reaction andmitochondrion serve as a principal source of it .Mitochondrion localization and function of heme oxygenase1in cigarette smoke-induced cell death in mammals has alsobeen reported, as HO1 works against cellular apoptosis(Slebos et al. 2007). Till date, we found that HO1 shows samekind of behavior in plants and animals; so here, we found that inplants also, HO1 shows its existence in mitochondrion. Wehave carried out localization experiment to subcellular fractiondesigned to determine the amount and nature of HO1 activitypresent in mitochondrion and chloroplast from green leaves ofsoybean (Glycine max). So here, we report the co-localizationof HO1 in chloroplast as well as mitochondrion.

Material and method

Subcellular fractionation

We have used centrifugal fractionation procedure described byPierpoint (1959). Experiment was conducted to obtain threefractions designated as chloroplast, mitochondrion, and solublefraction. Ten grams of soybean leaves, cut into pieces, and 50 mlof extracting medium (0.15 M phosphate, 0.3 sucrose, and0.01 M cysteine with pH adjusted to 7.5 was macerated for5 min using a plastic spatula and an ice-cold mortar. The slurrywas filtered through cheesecloth and the filtrate was centrifugedfor 2 min at 200×g . The sediment contained cells and cell debriswas discarded. The 200×g supernatant was re-centrifuged for7 min at 1,000×g . The sediment formed was resuspended in20 ml of the extracting medium and designated as chloroplasts.The 1,000×g supernatant was re-centrifuged for 15 min at 13,000×g . This sediment was resuspended in 20 ml of extractingmedium and designated mitochondria. The 13,000×gsupernatant was designated the soluble fraction. All operationswere carried out at 2 to 3 °C. The two fractions chloroplast andmitochondrion were assayed for heme oxygenase1 activity only.

Method for mitochondrial fraction purification

To prepare chloroplast-free mitochondria, we followed themethod of Bergman et al. (1980). The preparation medium wascomposed of 0.3 M sucrose, 1 mm EDTA, 1 mM MgCl2,25 mm Hepes (pH 7.8), 0.1 % (w/v) BSA (defatted), 4 mMcysteine, and 0.6 % (w/v) insoluble PVP (acid washed). After ashort homogenization followed by filtration, chloroplasts andcell debris were sedimented by centrifugation at 5,000 g for2min. Themitochondria in the supernatant were then spun downat 20,000 g for 2.5 min. Then two phase system was made byPEG. The phase system contained 5.8 % (w/w) dextran 500

batch 2836 (molecular weight 500,000), 5.8% (w/w) PEG 4,000(Mn 3,000–3,700), 5 mmol/kg K-phosphate (pH 7.8), 2 mmol/kgKCI, and 0.3mol/kg sucrose. Both phase systems give similarseparation of mitochondria and thylakoid membranes. The crudemitochondrial pellet was suspended in top phase (phase system,see below), bottom phase was added, and the phases were mixedand then separated by a short centrifugation. The top phasecontaining mostly thylakoid membrane fragments waswithdrawn and the bottom phase was repartitioned once or twicewith fresh top phase. The extracted bottom phase and theinterface were diluted eight times with preparation mediumwithout cysteine and PVP and centrifuged at 5,000 g for2 min. Finally, the mitochondria were collected from thesupernatant by centrifugation at 1,000 g for 10 min. The pelletobtained after phase partition was suspended in 0.25 M sucrose,1 mm KH2PO4, 10 mm Hepes (pH 7.2), and 0.1 % (w/v) BSA(defatted). About 2 ml of this suspension was layered on top of adiscontinuous Percoll gradient. The gradient was prepared in 12-ml cellulose nitrate tubes by layering 2 ml 60 % (v/v) Percoll,3 ml 27 % (v/v) Percoll, and 2 ml 21% (v/v) Percoll. In additionto Percoll, all layers contained 0.25 M sucrose, 1 mm KH2PO4,10 mM Hepes (pH 7.2), and 0.1 % (w/v) BSA (defatted). Thegradient was centrifuged at 10,000 rpm (9,000 gmax) for 30minin a Beckman 40 rotor (fixed angle) using a Beckman L2-65 Kultracentrifuge. After centrifugation, the mitochondrial band, onthe interface between the 27 (v/v) and 60 % (v/v) Percoll layers,was taken out by carefully pipetting.

Enzymatic assay

We have followed the method of Yannarelli et al. (2006). Hemeoxygenase activity was assayed as previously described withminor modifications. The assays (1 ml final volume) contained250 μl of chloroplast and mitochondrion extract (0.5 mgprotein), 10 μM hemin, 0.15 mg ml−1 bovine serum albumin,50 μg ml−1 (4.2 μM) spinach (Spinacia oleracea) ferredoxin(Sigma Chemical Co.), and 0.025 units mL−1 spinachferredoxin-NADP+reductase (Sigma Chemical Co.). Thereaction was started by adding NADPH to a final concentrationof 100 μM, samples were incubated at 37 °C for 30 min, andBV formation was calculated using the absorbance change at650 nm. The concentration of BV was estimated using a molarabsorption coefficient at 650 nm of 6.25 mM−1 cm−1 in 0.1 MHepes-NaOH buffers (pH 7.2). One unit of the enzyme forms1 nmol of biliverdin in 30 min under assay conditions.

Chloroplast and mitochondrial DNA isolation

Chloroplast and mitochondrial DNA has been isolatedaccording to Triboush et al. (1998). Green seedlings of soybean,kept in the dark for 48 h, are harvested, cooled at 0 °C, andground in an extraction buffer (containing 400 mM sucrose,50 mM Tris pH 7.8, 20 mM EDTA-Na2, 0.2 % BSA, 0.2 %

S. Dixit et al.

βME). The homogenate is filtered through dense nylon andcentrifuged at 200×g . The supernatant is then re-centrifuged at3,700×g . Finally, the organelle pellet is washed and precededfor lysis and purification to isolate the DNA using sodiumdodecyl sulphate (SDS) and chloroform: phenol methodsubsequently. Then DNA is precipitated using ammoniumacetate and isopropanol. Precipitated DNA is sequentiallywashed with ethanol, dried at RT, and redissolved in TE buffer.

Transcriptional level analysis

Semiquantitative RT-PCR has been performed to analyze HO1transcript level analysis in chloroplast and mitochondrion.Primers 5′-GATCCCCAAGCATTCATTTG-3′ and 5′-TTTCTTCCAGACAGTGGTCC-3′ were designed to amplifythe 476–701 bp region of the G . max HO1 DNA fromchloroplast and mitochondria (GenBank accession no.AF320024). The cycle number for PCR was 35. To standardizethe result, relative abundance of actin was determined and usedas internal standard. Aliquots from the PCR were loaded on1.2 % agarose gels with the use of ethidium bromide.

Western-blot analysis for HO1

Homogenates of organelles obtained for HO activity assayswere also analyzed by Western immunoblot technique. Fortymicrograms of protein from organelles homogenates weresubjected to SDS-polyacrylamide gel electrophoresis using a12 % acrylamide resolving gel (Mini Protean II System, Bio-Rad, Hertz, UK), according to Laemmli (ref from salt,Balanstrasse). Separated proteins were then transferred tonitrocellulose membranes and nonspecific binding ofantibodies was blocked with 3 % nonfat dried milk in PBS,pH 7.4 for 1 h at room temperature. Membranes were thenincubated overnight at 4 °C with primary antibodies raisedagainst Arabidopsis thaliana HO1, diluted 1:2,000 in Tris-NaCl buffer plus 1 % nonfat milk. Immune complexes weredetected using peroxidase-conjugated goat anti-rabbitimmunoglobulin G and visualized with the enhancedchemiluminescence. The films were scanned.

Results

Organelle isolation

The procedure described in material and method has been usedsuccessively for isolation of mitochondria and chloroplast.Subcellular partially purified fraction of chloroplast andmitochondrion were prepared from leaf samples. We haveoptimized the isolation procedure to get the best yield oforganelle and their DNA. HO1 activity has been analyzed inboth the fractions.

HO1 biochemical analysis

Data in Fig. 1 shows HO1 activity in chloroplast andmitochondrion. We have found 48 % higher activity of HO1in chloroplast in comparison to mitochondrion (Table 1). Ontreatment with toxic concentration of CdCl2 which is aninducer of ROS species, we have found 2.73-fold increase inHO1 activity in mitochondrial fraction (Table 1).

Organelle DNA isolation

The DNA preparations were successfully completed withconcentrations around 500 ng/μl. Samples were free fromgenomic DNA contamination as we have given optionalDNAse treatment. Organelle DNA obtained from leaves ofGlycine max has shown the expected pattern of restriction.Here, we have shown the restriction digestion of chloroplastand mitochondrial DNAwith ECOR1 (Fig. 1).

Semiquantitative RT and Western blot analysis

Level of HO1 enzyme in organelles has also been analyzedthrough semiquantitative RT-PCR (Fig. 2). Transcript level ofHO1 in partially purified DNA sample of mitochondria andchloroplast has been measured. Transcript level of HO1

Fig. 1 Ethidium bromide stained agarose gel of electrophoreticallyseparated sunflower chloroplast DNA and mitochondrial DNA restrictionfragments. Lane 1 cpDNA, undigested chloroplast DNA; Lane 2mtDNA, undigested mitochondrial DNA; Lane 3 , chl DNA/EcoR I,chloroplast DNA digested by EcoR I; mtDNA/EcoR I, mitochondrialDNA digested by EcoR I. Samples were size fractionated on 1 % agarosegels containing 0.1 M Tris-acetate (pH 8.0) and 1 mM EDTA.Electrophoresis was for 16 h at 1 V/cm

In vitro evaluation of mitochondrial–chloroplast subcellular

was found higher in chloroplast in comparison tomitochondria due to its higher activity in chloroplast.We have found a positive correlation with biochemicalanalysis. To further examine the subcellular localizationof HO1 protein, we conducted protein gel blot analysiswith HO1 antisera using subcellular fractions. The HO1protein at 31 kDa was detected in the chloroplast andmitochondrial fraction (Fig. 2). Protein band was moreintense in chloroplast fraction.

Discussion

As we have found in HO1 activity, we have reached tothis conclusion as to the localization of HO1 inmitochondrion due to electron donors of HO1-catalyzedreactions, NADPH, and ferredoxin provided by

mitochondria. This small amount of activity may bedue to occlusion or due to retention of small amount ofexternal membranes holding HO1. Where in chloroplasthas the higher activity due to retention of innermembrane due to much higher level of minerals andproteins found in chloroplast. Leaf mitochondria containonly a small percentage of the HO1 activity present inthe leaf, but its distribution within mitochondria appearsto resemble that seen in animal tissues (Slebos et al.2007). HO1 activity increases on generation of ROSspecies in mitochondrion on Cd-induced oxidative stress.We have found correlative result on transcriptional levelanalysis. We can also suggest that HO1 might be locatedin the outer membrane of chloroplast from where it canbe easily translocated to mitochondria to provide energythrough NADP-dependent ferredoxin.

Immunoblot analysis showed that mitochondria alsocontain detectable level of HO1 protein. A positive correlationhas been found in biochemical activity transcript level andprotein expression level. This also suggest that transit peptideof HO1 is functional and sufficient to transport it fromchloroplast and mitochondria. High level of HO1 protein liesin a good agreement with the observation that hemeoxygenase activity is localized in plastids (Cornejo et al.1998), but a little controversial HO1 activity was also foundin mitochondria.

The question of the intracellular localization andorganization of heme-oxygenase enzyme was first raised byDavis et al. (2001). Finding of its localization in chloroplastdue to its role in phytochrome synthesis has been achieved(Muramoto et al. 1999). Later on, researchers have supportedthis observation, but here, we report that it is co-localized inmitochondria.

One strong reason of its existence in mitochondria is thatHO1 might be associated with the membrane, therefore mighthave membrane binding site as was previously suggestedbased on the finding of a heavy metal binding site onantioxidant enzyme (Smarrelli and Campbell 1983). An

Table 1 Heme oxygenase (HO1)activity in chloroplast andmitochondria

The HO1 activity in cellularorganelle with and without Cdtreatment of 200 μM. Valuespresented of three replicates andare statistically significantaccording to Tukey’s HSD test (P< 0.05), within individual Cutreatments. Text in blue shows theresult of another method ofmitochondria isolation

HO1 activity

μM (biliverdin)/min/μg( protein)

HO1 activity

μM (biliverdin)/min/μg( protein)

Mitochondria Chloroplast Mitochondria

Control 0.0014 0.00893 0.0020

0.0016 0.00855 0.0018

0.0017 0.00892 0.0017

Mean 0.0015±0.00015 0.0087±0.00021 0.0018±0.00012

Cd treatment 0.0039 0.00885 0.0032

0.0041 0.00891 0.0045

0.0043 0.00891 0.0038

Mean 0.0041±0.0002 0.00889 ±0.00014 0.0038±0.00016

Fig. 2 a Transcript level of HO1 has been measured in chloroplast andmitochondrial DNA. Actin gene is used as an internal standard. bSpecificity of the HO1 antibody. Organelles samples were separated bySDS-PAGE, blotted to nitrocellulose, and probed with HO1 antibody, asdescribed in “Materials and methods.” The weak band of 32 kDa is alsodetected in mitochondria

S. Dixit et al.

important enzyme of heme biosynthesizing pathway isprotoporphyrinogen oxidase. This enzyme has been found intwo isoforms as follows: a plastidic and a mitochondrialisoforms. Existence of heme synthesizing pathway gives astrong support for the presence of heme degrading enzyme(heme oxygenase) in mitochondria along with chloroplast.

In addition, there is a possibility that for regulation ofsynthesis and turnover of hemin degradation reaction, HO1is probably co-localized in chloroplast and mitochondria.After considering all these facts, we justify our observationof co-localization of HO1.

Acknowledgment Gyan Singh Shekhawat gratefully acknowledgesfinancial support received from the Science andEngineeringResearchBoard(SERB), New Delhi, India under young scientist scheme.

Conflict of interest Authors do not have any conflict of interest

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In vitro evaluation of mitochondrial–chloroplast subcellular