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Plant Physiol. ( 1985) 78, 115-1200032-0889/85/78/011 5/06/$0 1.00/0

Iron Deficiency Decreases Suberization in Bean Roots through aDecrease in Suberin-Specific Peroxidase Activity1

Received for publication October 29, 1984

PETER C. SIJMONS*, P. E. KOLATTUKUDY, AND H. FRrrs BIENFAITDepartment ofPlant Physiology, University ofAAmsterdam, Kruislaan 318,1098SM Amsterdam, Netherlands (P.C.S., H.F.B.); and Institutefor Biological Chemistry, WashingtonState University, Pullman, Washington 99164-6340 (P.E.K)

ABSTRACT

The suberin content of young root parts of iron-deficient ad iron-sufficient Phaseolus valgaris L cv Prelude was determined. The aliphaticcomponents that could be released from subern-enriched fractious byLiAID4 depolymerization were identified by gas chromatogrphy-mssspectrometry. In the normal roots, the major aliphatic componets werew-hydroxy acids and dicarboxyli acids in which saturated CI, admonounsaturated Cis were the dominant homologues. Iron-deficient beanroots contained only I I% of the aliphatic component of suberin foudin control roots although the relaive composito of the constitu s wasnot significantly affected by iron deficiey. Analysis of the ticcomponents of the subenn polymer that could be released by alkalinenitrobenzene oxidation of bean root samples showed a 95% dease inp-hydroxybenzaldehyde, vanilin, and syringaldehyde under iron-defitconditions. The inhibition of suberin synthesis in bean roots was not dueto a decrease in Fe-dependent w-hydroxyhse activity since normalw-hydroxylation could be demonstated, both in vitro with microsomalpreparations and in situ by labeling of w-hydroxy and dicarboxylic acidswith l'4Cacetate. The level of the isozyme of peroxidase that is specifi-cally associated with suberization was suppressed by iron deficiency to25% of that found in control roots. None of the otber extted isozymesof peroxidase was affected by the iron nutritional status. The activity ofthe suberin-associated peroxidase was restored within 3 to 4 days afterappliation of iron to the growth medium. The results suggest that, inbean roots, iron deficency causes inhibition of suberization by causing adecrease in the level of isoperoxidase activity which is required forpolymerization of the aromatic domains of suberin, while the ablity tosynthesize the aliphatic components of the suberin polymer is not im-paired.

plasma membrane-bound redox system (22) which uses cytosolicNADPH as electron donor (23). Grasses do not reduce ferricchelates at appreciable rates, and also do not extrude protons asa response to iron starvation ( 18).

Kinetic studies of Fe3" reduction in iron-deficient bean rootsindicated earlier that, at least at low iron concentrations, anextracellular diffusion barrier influences the rate of this process(1). In addition to the cell wall, suberin may be a functional partof the diffusion barrier. This polymer, which is composed ofaliphatic and aromatic domains interspersd with wax layers(lI ), is deposited in the walls of epidermal and endodermal rootcells (13, 14, 16). In general, the major aliphatic components ofthe suberin polymer are long chain (C1626) w-hydroxy anddicarboxylic acids (12). These acids are thought to be involvedin cross-linking the aromatic matrix and constitute the aliphaticdomain of the polymer. Vanillin and p-hydroxybenzaldehydehave been found to be produced by alkaline nitrobenzene oxi-dation of the aromatic domains of suberin (7). Polymerizationof the aromatic monomers is thought to be mediated by peroxi-dase (2, 8) in a manner analogous to that suggested for ligninsynthesis (10).With the present knowledge of suberin synthesis, at least two

sites can be pointed out where iron deficiency may have an effect.(a) o-Hydroxylation of fatty acids which probably involves a CytP450 enzyme system (24). This hydroxylation can be inhibited invitro with the ferrous scavenger a,a-dipyridyl (24), indicating theinvolvement of loosely bound Fe. (b) A heme-Fe containingperoxidase specifically associated with suberin synthesis (8). Ac-cording to the working hypothesis concerning the structure andbiosynthesis of suberin, both enzymes are essential for the for-mation of the suberin polymer.

Iron deficiency during growth has pronounced effects on theanatomy and metabolism of roots in dicots and non-grass mon-ocots (5, 18). A high capacity for proton extrusion is developedin the epidermis (5, 19) which is correlated with the formationof rhizodermal transfer cells (18). Also, the Fe3" reduction activ-ity in epidermis cells is increased about 10-fold (6, 20). Reductionof Fe3' to Fe24 is an essential step in the process of iron uptakein dicotyledonous plants (6), and the reduction is mediated by a

'Supported in part by a grant PCM-8306835 from the NationalScience Foundation and by a travel grant (to P.C.S.) from the Foundationfor Fundamental Biological Research (BION), which is subsidized by theNetherlands Organization for the advancement ofPure Research (ZWO).Scientific Paper No. 6964, Project 2001, College ofAgricultural ResearchCenter, Washington State University, Pullman, WA 99164.

MATERIALS AND METHODS

Growth of Plants. Six-d-old etiolated seedlings of Phaseolusvulgaris L. cv Prelude (Royal Sluis, Enkhuizen, The Netherlands)were grown on nutrient solution with or without 40 yM FeNa-EDTA as described previously (23). Unless mentioned otherwise,bean roots were harvested 8 to 9 d after transfer to nutrientsolution. Seeds of Zea mays L. cv Capella (Van der Have,Kapelle, The Netherlands) were extensively rinsed with deionizedH20 and imbibed for 16 h in 0.05 mM CaSO.. The seeds weregerminated for 10 d in moistened perlite in a growth chamberand then transferred in sets of 24 to 5 L black plastic containerswith the following, aerated, nutrient solution: 2.2 mm K+, 0.5mM Ca2", 0.08 mM Mg2e, 0.5 mM NO3, 0.1 mM H2PO4, 0.08mM SO42-, 0.01 mM Na+, 9 jM Mn24, 0.7 juM Zn2", 0.3 jim Cul2,0. 12 jM Co2+, 120 jM Cl-, 45 uM BO3, 0.3 jM MoO42-. Fe wasadded as 0.1 mM (control plants) or 60 nM (iron-deficient plants)Fe3+NaEDTA. The Mn2+ concentration was lowered to 0.9 juM

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Plant Physiol. Vol. 78, 1985

in the iron-deficient nutrient solution (cf 25). The pH wasadjusted to 5.0 with KOH. The solutions were replaced withfreshly prepared nutrient solution every 3rd d. Three d aftertransfer to nutrient solution, the cotyledons were carefully re-moved to diminish fungal growth. The plants were grown in agrowth chamber (16 h light, 22C) under 43 w.m2 (Philips HPI/T) and 65% RH. Maize roots were harvested 3 weeks aftertransfer to nutrient solution.

Isolation of Roots. After rinsing the roots in ice-cold 0.1strength iron-free nutrient solution, young lateral and the young-est 3 to 5 cm of the main roots were carefully isolated with aforceps and kept on ice until all roots were harvested. Thenutrient solution was decanted and the roots were gently blotteddry and weighed. The isolated roots were then either used directlyfor in vivo labeling experiments, or homogenized with mortar,pestle, and quartz sand for preparation of a subcellular fractionaccording to (24). For suberin analysis or peroxidase extractions,the isolated young roots were homogenized in liquid N2 andlyophilized.

Analysis of Suberin. Portions of I g lyophilized root sampleswere Soxhlet extracted (48 h with CHC13 and 48 h with CH30H),ground to a powder (Wig-L-Bug amalgamator), and againSoxhlet extracted (24 h CHC13, 24 h CH30H). For analysis ofthe aliphatic components of the suberin polymer, the residuewas dried and 500 mg of this powder was depolymerized withLiAlD4, and the products were fractionated by TLC, derivatizedwith bis-N,O-trimethylsilyl acetamide, and analyzed by com-

bined GC-MS (Hewlett Packard 5840A/5985) as described pre-viously (27). For analysis of the aromatic components of thesuberin polymer, portions (about 700 mg) of the powdered andSoxhlet-extracted root samples were added to 20 ml 2 N NaOHand 1.0 ml nitrobenzene and heated in a stainless steel bomb at160°C for 3 h. The reaction mixture was cooled, filtered, andextracted with diethyl ether. The aqueous fraction was acidifiedand extracted with diethyl ether and the products in the etherfraction were subjected to TLC, acetylation, a second TLC, andGC-MS characterization (9).

In Vitro Assay for w-Hydroxylase Activity. Enzyme prepara-

tion (50-450 Mg microsomal protein) was added to test tubescontaining, in a total volume of 2 ml, 0.1M Tris-HCI buffer (pH7.5), 0.25M sucrose,1 mM MgCl2,1 mm ascorbate, 0.5 mM DTE,1 mm NADPH, 4.5 mM glucose-6P,I unit glucose-6P dehydro-genase, and 25MM[l-4CJpalmitic acid (54.2 Ci/mol, New Eng-land Nuclear) which was dispersed in water with the aid ofTween 20 and sonication. After incubation in a shaking waterbath for 60 min at30°C, the reaction was stopped with theaddition ofI ml N HCI. The lipids were extracted, reducedwith LiAIH4, and the C16-diol fraction was isolated as describedpreviously (24).w-Hydroxy products were separated from prod-ucts of competing oxidations with HPLC on a Waters NovapacC18 column (17% water in methanol, isocratic solvent system,flow rate 2 ml min-') coupled to a radioactivity flow detector(Flow-One HS, Radiomatic Instruments & Chemical Comp.).Authentic hexadecane-1,16-diol and hexadecane-1,2-diol were

used as standards.In Situ w-Hydroxylation. Isolated young roots (2 g fresh

weight) were incubated forI h at27°C inI mM Mes, in iron-

free nutrient solution (pH 5.0), and 12Mm [2-'4C]acetate (54Ci/mol, New England Nuclear). The roots were then decanted andrinsed several times with cold water, homogenized in liquid N2,and lyophilized. The dry root material was Soxhlet extractedwith CHC13 for 24 h. The chloroform-soluble material was

reduced withULAIH4 and fractionated on TLC (24). The silicagel was scraped off in portions of 10 mm, added to TolueneScintillator (Packard), and assayed for14C. RF values were com-pared with those ofapple cutin hydrogenolysate components(24).

Suberin Synthesis in Epidermis. Isolated youngest parts of themain roots (i.e. unbranched root parts) were incubated for 2 hin [2-'4C]acetate as described above. After decanting and washingthe roots, they were kept on ice while epidermal layers werestripped off with fine surgical forceps. Only samples that showedclear separation from endodermal tissues were retained for fur-ther analysis. The collected epidermal samples were gently blot-ted dry with tissue paper and weighed. After adding unlabeledroots as carrier material, the samples were frozen, lyophilized,and homogenized in a Teflon container (precooled in liquid N2)with a Braun Mikro-dismembrator II. The resulting powder was

Soxhlet extracted (48 h CHC13 and 48 h CH30H), dried at 80C,and depolymerized with LiAlH4. The released aliphatic mono-mers were extracted with chloroform, concentrated, dried underN2, and counted in a liquid scintillation counter.

Extraction and Assays of Root Peroxidases. Lyophilized rootsamples (200 mg) were suspended in 10 ml 0.04 or 1.0 MK-acetate buffer (pH 4.3) and ground in a Ten Broeck homoge-nizer at0°C. The resulting slurry was filtered through four layersof cheesecloth and the filtrate was centrifuged at 50,000g for 30min. The supernatants were dialyzed against 2x 5 L 0.01 M

K-acetate (pH 4.3) and lyophilized. The residues were resus-

pended in 200 to 400,l 0.04M K-acetate buffer to obtain equalprotein concentrations, centrifuged for 5 min in an Eppendorfcentrifuge, and 2 to 8 Ml of these supernatants were used for gelelectrophoresis. Isozymes were separated on continuous 6% poly-acrylamide gel with a LKB 2117 Multiphore apparatus at0°C(3). The gels were stained for peroxidase activity with 3-amino-9-ethylcarbazole and H202, fixed, and scanned as describedpreviously (8). Total peroxidase activities in crude extracts were

determined, on aliquots taken before the dialysis step, by meas-

uring guaiacol oxidation (26). Protein was determined accordingto Bradford (4), using bovine albumin as standard.

RESULTS

Analysis of Suberin in Iron-Deficient and Control Bean Roots.Bean roots were extensively extracted with organic solvents andanalyzed for suberin content by measuring the aliphatic com-

ponents released by reductive depolymerization with LiAlD4,and the aromatic components released by alkaline nitrobenzeneoxidation. The composition of the aliphatic components of thesuberin polymer is given in Table I. The major aliphatic com-

ponents were w-hydroxy acids and dicarboxylic acids. In bothgroups, saturated C16 and monounsaturated C18 were the domi-nant components, and homologues with even numbers of carbonatoms up to C24 were found in both cases. Very long chain fattyacids and fatty alcoholsup to C28 were also found. Roots sub-jected to iron deficiency released only190,Mg aliphatic monomersper gram of extracted tissue compared to 1690ug from controlroots. In spite of the severe inhibition caused by iron deficiency,the relative proportion of the various aliphatic components was

not altered (TableI).Since the amount of p-hydroxybenzaldehyde and vanillin re-

leased by nitrobenzene oxidation of suberin polymers from othertissues appeared to be a reasonable measure ofsuberin (7), similaroxidation was attempted on the present root samples. p-Hydrox-ybenzaldehyde was the major aromatic aldehyde released fromthe polymeric materials from the control roots and smalleramounts of vanillin and syringaldehyde were also found (TableII). The polymeric material from Fe-deficient roots released only5% of the aromatic aldehydes generated from control roots(Table II).

Suberin Synthesis in Bean Root Epidermis. To establishwhether in very young root tissue suberin is synthesized in theepidermis, a suberin fraction was derived from epidermal tissuethat was isolated after incubation in['4C]acetate. After depoly-merization of this labeled suberin fraction, the radioactivity was

116 SIJMONS ET AL.

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IRON DEFICIENCY AND SUBERIN SYNTHESIS

Table I. Aliphatic Composition ofthe Suberin Polymersfrom Iron-Deficient and Control Roots ofP. vulgaris

Values given in parentheses are the percentages of each group ofmonomers.

Control Iron-DeficientRoots Roots

,g/g dry tissue wtFatty Alcohols

C18:i 2.9 0.2C20 5.4 5.3C22 37.2 1.6C24 15.3 0.8C26 0.2 0.1C28 2.2Total 63.2 (3.7) 8.0 (4.2)

Fatty AcidsC18g: 3.7 0.4C20 5.7 10.3C22 29.2 2.9C24 43.4 2.3C26 1.4 0.1Total 83.4 (4.9) 16.0 (8.5)

c-Hydroxy AcidsC,6 381.9 38.1C18g: 249.6 24.6C,8 98.7 5.0C20 191.0 6.0C22 55.9 0.9C24 13.5Total 990.6 (58.6) 74.6 (48.7)

Dicarboxylic AcidsC,6 234.1 24.3Ci8g: 173.4 24.6C,8 63.0 5.09,10-Dihydroxy C,8 7.1 3.6C20 6.0C22 22.8 0.9C24 4.4Total 504.8 (30.0) 64.4 (33.9)

Polar Acids9,10,18-Trihydroxy C,8 6.6 1.9Total 6.6 (0.4) 1.9 (1.0)

Table II. Amounts ofp-Hydroxybenzaldehyde, Vanillin, andSyringaldehyde That Could Be Released by Nitrobenzene Oxidation of

Iron-Deficient and Control Bean Roots


,ug/g dry tissuep-Hydroxybenzaldehyde 58.0 2.3Vanillin 14.0 0.4Syringaldehyde 12.5 0.8

Total 84.5 3.5

determined in the released aliphatic monomers. Samples fromcontrol and iron-deficient roots contained 28,530 and 15,770dpm/g fresh weight epidermal tissue, respectively. These resultsindicated that suberin was indeed synthesized in the epidermisof very young roots.

o-Hydroxylation of Fatty Acids in Bean Roots. The in vitrocapacity of the w-hydroxylation enzyme system was tested withextracts of iron-deficient and control bean roots. ['4C]Palmiticacid and NADPH were added to microsomal preparations fromthe roots. After the incubation, lipids were extracted from the

reaction mixture and the formation of labeled w-hydroxy pal-mitic acid was determined after reduction with LiAlH4 andseparation of an alkane diol fraction by TLC. The diols wereidentified with radio-HPLC. Both the yield of microsomes (datanot shown) and the specific activity of o-hydroxylase (Fig. 1)were increased by iron deficiency of the roots.The in situ w-hydroxylase activity was assessed by incubating

young isolated roots in ['4C]acetate. Chloroform-soluble lipidsfrom these roots were reduced with LiAlH4 and separated byTLC. The amount of radioactivity that was recovered in the diolfraction as a percentage of total label in the lipid fraction wasnot significantly changed by iron deficiency (Table III).

Peroxidase Activity in Bean Roots. Earlier results have indi-cated that a peroxidase is involved in the polymerization of thephenolic monomers for the aromatic domains of the suberinmatrix (8). We investigated the activities of root isoperoxidasesin lyophilized root samples. Roots were extracted with K-acetate(0.04 M, pH 4.3), the extracts were dialyzed, concentrated, andsubjected to PAGE, followed by enzymic staining (Fig. 2). Equalamounts of total protein were placed in each slot, so relativespecific activities can be compared directly from the intensity of

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P9 microsomal proteinFIG. 1. Rate of w-hydroxylation of palmitic acid by a microsomal

preparation from iron-deficient (-Fe) and control (+Fe) bean roots atdifferent protein concentrations. Reaction mixtures contained I mMNADPH and its regenerating system and 25 uM ['4C]palmitic acid in atotal volume of 2 ml buffer as described in "Materials and Methods."The reaction mixtures were incubated for 60 min at 30°C.

Table III. In Situ Rate ofw-Hydroxylation in Young Iron-Deficientand Control Bean Roots

After incubation of isolated roots in ['4C]acetate, chloroform-solublelipids were reduced with LiAlH4 and separated on TLC. w-Hydroxylationproducts are recovered as diols.


Total dpm on TLC 1,098,800 901,600dpm in alcohol fraction 999,900 831,700dpm in diol fraction 59,000 37,800dpm in diol fraction as % of total 5.4% 4.2%

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Plant Physiol. Vol. 78, 1985

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FIG. 3. Development of peroxidase activity in young bean roots dur-ing growth on iron-free (-Fe) or iron-containing (+Fe) nutrient solution.Eight d after transfer to iron-free nutrient solution, 40 AM FeEDTA wasadded to the nutrient solution (arrow).

solution, the specific total peroxidase activity in extracts fromiron-deficient roots leveled off at about 10 units/mg proteinwhile the activity in extracts from control roots continued toincrease (Fig. 3). Addition of 40 Mm FeEDTA at the 8th d toiron-deficient plants resulted in restoration of root peroxidaseactivity to the control levels in the following 3 to 4 d (Fig. 3).

Analysis ofSuberin in Iron-Deficient and Control Maize Roots.As a representative of the grasses, a class of species that does notrespond to iron deficiency with increased Fe3" reduction activity(21) or increased proton extrusion (18), maize roots were usedfor suberin analysis. Unlike in bean, maize roots demonstratedsevere growth inhibition under iron-deficient conditions, but theiron status of the plant was of little influence on the suberizationof the maize roots. The total amounts of aliphatic monomersthat could be released from control and iron-deficient roots were132 and 190 tg/g solvent-extracted root tissue, respectively.

.i,_.

FIG. 2. Effect of iron deficiency on isoperoxidases of young beanroots. Isoperoxidases were extracted from young lateral and main roots8 d after transfer to iron-free (-Fe) or iron-containing (+Fe) nutrientsolution. The density scan was made after staining for peroxidase activitywith 3-amino-9-ethylcarbazole. Arrow indicates the position of the an-

ionic peroxidase which activity was most susceptible to iron deficiency.

the staining. Ofall peroxidase bands, only one anionic peroxidaseshowed approximately 75% less activity in extracts from iron-deficient roots compared to extracts from control roots (Fig. 2).The other isoperoxidases were hardly influenced by the iron-status of the plants (Fig. 2). Similar results were obtained afterextraction with a buffer of high ionic strength (I M K-acetate,pH 4.3) to release cell wall-bound isoperoxidases; only oneanionic peroxidase showed a severe decrease during iron defi-ciency (data not shown).

Figure 3 shows the development of peroxidase activity in theroots during the growth of plants on iron-free and control nutri-ent solutions. During the first 8 d after transfer to nutrient

DISCUSSION

In bean plants, iron deficiency is accompanied by a severeinhibition of suberin synthesis in the roots. The content of boththe aliphatic and the aromatic components of suberin show adecrease of at least 90%, while the appearance of the roots andtheir growth seem to be normal.Two iron-containing enzymes which are essential for suberin

synthesis were tested for their activity. The activity of the P450containing enzyme system for the o-hydroxylation of fatty acidswas not impaired by iron starvation, both in vitro with micro-somal preparations from young roots, and in situ with intactyoung roots. The high c-hydroxylase activity that was present inmicrosome preparations from iron-deficient roots compared tocontrol preparations (Fig. 1) may be the result of an increase inrelative amounts of ER vesicles in the microsomal fraction.Under iron deficiency, a substantial increase in ER is observedin a number of species (18). The results ofthe in vitro and in situco-hydroxylase measurements show that, while the incorporationof c-hydroxy and dicarboxylic acids into the suberin polymerwas severely inhibited in the iron-deficient roots, the ability tosynthesize these aliphatic components was not inhibited.The suberin-specific peroxidase showed a severely decreased

activity in extracts from iron-starved bean roots compared tocontrol roots. When iron-deficient plants were transferred to

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IRON DEFICIENCY AND SUBERIN SYNTHESIS

normal iron-containing nutrient solution, the extractableamount of this peroxidase restored to the normal levels within 3to 4 d. We suggest that the inhibition of suberin synthesis iscaused by a lowered activity of the suberin-specific peroxidase.

Roots of iron-deficient plants are known to accumulate lowmol wt phenolic compounds (5, 17). At equal rates of phenolicprecursor supply in iron-deficient and control roots, decreasedsuberization would result in an accumulation of aromatic pre-cursors. Assuming that the suberin is composed of 5 to 10%aliphatics and 40 to 45% aromatics ( 12), the amount ofaromaticprecursors accumulating in iron-deficient roots could be as highas 7 mg chlorogenic equivalents per g dry tissue (before solventextraction). This is in the order reported for iron-deficient peanutroots ( 17).Other types of stress, e.g. wound healing (2, 7) and Mg defi-

ciency (14), enhance suberin synthesis. The present finding is thefirst example where a stress situation results in a decreasedsuberin synthesis. In contrast, in maize roots there was noapparent influence of iron nutrition on suberin content. How-ever, iron deficiency caused a substantial retardation of rootgrowth in these plants. It is therefore difficult to assess whetheriron shortage affects the synthesis of suberin, directly via theactivities of iron-containing enzymes or indirectly via the rate ofroot growth.

In dicotyledonous plants, iron deficiency causes a pronouncedaccumulation of reducing equivalents in the root epidermis (5).This excess can be used for the reduction of ferric ions in thenutrient solution via an enzyme system in the root plasmamembrane that transfers electrons from cellular NADPH toextracellular acceptors (23). How iron starvation leads to theaccumulation of reducing power is unknown. Two of us (23)proposed that when synthetic processes involving iron-contain-ing enzymes are inactivated by iron deficiency, an accumulationof reducing power may result. Suberin synthesis takes place inthe epidermis (and endodermis) ofyoung roots, and is susceptibleto inhibition by iron deficiency. Thus, in principle, reducingequivalents not used for suberin (fatty acid) synthesis could beused for the reduction of extracellular ferric chelates, ifNADPHproduction remained unchanged. It can be calculated from TableI and Reference 20 that the amounts of NADPH necessary forthe synthesis of the quantities of suberin present in normal beanroots are an order of magnitude smaller than the amounts ofreducing equivalents that are available, via NADPH, for Fe3+-chelate reduction under iron deficiency. The inhibition of suberinsynthesis is therefore not sufficient to explain the increased Fe3"reduction activity of iron-deficient bean roots.On the other hand, inhibited suberin synthesis could be ben-

eficial to iron-stressed roots. The zones in the roots where ferricreduction can take place, normally about 0.5 to 1 cm in beanroots, are elongated to several cm under iron deficiency (20).This means that the root cells retain their capacity to reduce Fe3"for a longer period of time. Inhibition of deposition of a suberinlayer at the epidermis probably plays a role in providing thelonger region for Fe3" reduction.

This report shows that the typical response of dicotyledonousplant roots to iron deficiency, i.e. accumulation ofNADPH (23),synthesis of rhizodermal transfer cells (18), and proton extrusion(5), become apparent when iron starvation has not yet affectedthe activity of all iron-containing enzymes. The w-hydroxylationsystem, which reportedly contains a loosely bound Fe (24), isactive as shown in the present paper. The heme enzymes show adiverse response: catalase is reduced to 60% (20), the suberin-specific peroxidase to about 25% (Fig. 2), while the other extract-able peroxidases are hardly affected (cf 15). Cyt P450 in thew-hydroxylation system, mitochondrial respiration, and the non-heme iron protein proline hydroxylase (Sijmons, unpublished)are also unimpaired by the degree of iron stress imposed on our

bean plants.Assuming that the peroxidases obtain their heme from a

common pool, the differential response ofheme enzymes to irondeficiency indicates differential effects on the synthesis of theapoprotein moiety of some enzymes. Another possibility is thatnot all the apoproteins have the same affinity for heme and,therefore, a lowered cellular heme level would first affect thoseenzymes with the higher Km for heme.The structure of suberin is poorly understood. According to

the working hypothesis concerning suberin structure, an aro-matic domain is attached to the cell wall using an isoperoxidaseand aliphatic domains are attached to this aromatic matrix. Thepresent finding that suppression of the isoperoxidase, postulatedto be involved in the synthesis of the aromatic matrix (2, 3, 7,8), by iron deficiency inhibits deposition of both the aliphaticand aromatic domains of suberin supports the above hypothesisconcerning suberin structure.

Acknowledgments-We thank Drs. C. L. Soliday and K. E. Espelie for theirtechnical assistance and advice.

LITERATURE CITED

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7. COTTLE W, PE KOLATTUKUDY 1982 Biosynthesis, deposition and partialcharacterization of potato suberin phenolics. Plant Physiol 69: 393-399

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10. GRISEBACH 1981 Lignins. In EE Conn, ed, The Biochemistry of Plants, Vol 7.Academic Press, New York, pp 457-478

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12. KOLATrUKUDY PE 1981 Structure, biosynthesis, and biodegradation of cutinand suberin. Annu Rev Plant Physiol 32: 539-567

13. PETERSON CA, RL PETERSON, AW ROBARDS 1978 A correlated histochemicaland ultrastructural study of the epidermis and hypodermis of onion roots.Protoplasma 96: 1-21

14. POZUELO PM, KE ESPELIE, PE KOLATTUKUDY 1984 Magnesium deficiencyresults in increased suberization in endodermis and hypodermis of cornroots. Plant Physiol 74: 256-260

15. PRICE CA 1968 Iron compounds and plant nutrition. Annu Rev Plant Physiol19: 239-248

16. ROBARDS AW, SM JACKSON, DT CLARKSON, J SANDERSON 1973 The structureof barley roots in relation to the transport of ions into the stele. Protoplasma77: 291-311

17. ROMHELD V 1979 Mechanismus der Aufnahme und Verlagerung von Eisen-chelaten bei hoheren Pflanzen. PhD thesis. Technische Universitat, Berlin

18. ROMHELD V, D KRAMER 1983 Relationship between proton efflux and rhizo-dermal transfer cells induced by iron deficiency. Z Pflanzenphysiol 1 13: 73-83

19. ROMHELD V, C MULLER, H MARSCHNER 1984 Localization and capacity ofproton pumps in roots of intact sunflower plants. Plant Physiol 76: 603-606

20. SIJMONS PC, HF BIENFAIT 1983 Source of electrons for extracellular Fe(III)reduction in iron-deficient bean roots. Physiol Plant 59: 409-415

21. SiJMoNs PC, FC LANFERMEIJER, HF BIENFAIT 1984 Root NAD(P)H levels inmonocotyledonous species differing in their response to iron-deficiency.Plant Physiol 75: S-193

22. SIJMONS PC, FC LANFERMEIJER, AH DE BOER, HBA PRINs, HF BIENFAIT 1984Depolarization of cell membrane potential during trans-plasmamembraneelectron transfer to extracellular electron acceptors in iron-deficient roots ofPhaseolus vulgaris L. Plant Physiol 76: 943-946

23. SIJMONS PC, W VAN DEN BRIEL, HF BIENFAIT 1984 Cytosolic NADPH is the

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120 SIJMONS ET AL. Plant Physiol. Vol. 78, 1985

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24. SOLIDAY CL, PE KOLATTUKUDY 1979 Biosynthesis of cutin. w-Hydroxylation IAA oxidase during tomato fruit development. J Food Sci 47: 158-161of fatty acids by a microsomal preparation from germinating Vicia faba. 27. WALTON TJ, PE KOLATTUKUDY 1972 Determination of the structure of cutinPlant Physiol 59: 1 1 16-1121 monomers by a novel depolymerization procedure and combined gas chro-

25. SOMERS 11, JW SHIVE 1942 The iron-manganese relation in plant metabolism. matography and mass spectrometry. Biochemistry 11: 1885-1897



iron deficiency decreases suberization in beanrootsthrough ... · containing, in atotal volumeof2...

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