Plant Physiol. (1982) 69, 0268-02720032-0889/82/69/0268/05/$00.50/0

Cytoplasmic Male Sterility in Barley'VIII. LIPOXYGENASE ACTIVITY AND ANTHER AMINO NITROGEN IN THE msml-Rfmla SYSTEM

Received for publication December 12, 1980 and in revised form Auguist 15, 1981

HANNU AHOKASDepartment of Genetics, University of Helsinki, P. Rautatiekatu 13, 00100 Helsinki 10, Finland

ABSTRACT

The lipoxygenase (LOX) activity was determined in almost isogenictypes of barley (Hordeum vulgare L.): normal cv. Adorra, cytoplasmic malesterile (msml), and msmi barley with restored fertility, heterozygous forthe Rfmila restorer gene. The LOX activity was lowest in male steriles inthe leaf tissue studied at the anthesis stage. TheLOX activity in developinganthers was higher than in leaf tissue, and decreased during degenerationof the sterile anthers.On polyacrylamide gel electrophoresis slabs, the LOX of anther homog-

enates moved in a complex which evidently carried some lipid and pigment,too. The LOX zones showed pseudoisoenzymic movement, Le. a gradualincrease in mobility dependent on the age of the anthers. On sodiumdodecyl sulfate-polyacrylamide gel electrophoresis slabs, the LOX zonescontained three polypeptides. When sporopolienin production ends in thefertile anthers, a fourth polypeptide (molecular weight 91,200) appears intheir LOX zones. This 'late' polypeptide is missing from steriles, and issuggested as being associated with the termination of sporopollenin pro-duction in the tapetum of fertiles.

Sterile anthers were found to be almost devoid of soluble NH2-N, whichsupports the idea of their starvation. This starvation can reasonably beheld responsible for the absence of late proteins (e.g. the 91,200 daltonpolypeptide), and is reinforced by the uncontrolled production of sporopol-lenin.

Cytoplasmic male sterility, or male sterile maternal 1 (msml),was found as a natural variant in an Israeli strain of the wildprogenitor of barley (Hordeum vulgare ssp. spontaneum [C. Koch]Thellung) (2). The original strain carries a dominant fertilityrestorer gene designated as Rfmla. A number of restorer genes, aswell as partial restorer genotypes, have been found in wild barleystrains from Israel (4, and unpublished). These additional restorergenes appear in male fertile cytoplasms. This finding and pecu-liarities in their geographic distribution suggest that the restorergenes are responsible for special ecophysiological adaptations.Another, different type of maternal male sterility (msm2) wasrecently found by the author (in preparation).LOX2 activity in leaf and anther tissue in various, near-isogenic

derivatives ofmsml and normal barley is described in this paper.The interest in LOX activity takes two forms. First, LOX is locatedeither entirely in plastids (12, 13), or else partly in mitochondria(28) and may, therefore, be associated with maternal inheritance.Second, LOX may be associated with the production of sporopol-

'The work was done under the auspices of the Research Council forAgriculture and Forestry of the Academy of Finland.

2Abbreviations: LOX, lipoxygenase, linoleate: oxygen, oxidoreductaseEC 1. 13.11.12; PAGE, polyacrylamide gel electrophoresis.

lenin in the tapetum. Sporopollenin is evidently an oxidativepolymer of carotenoids or carotenoid esters (8, 9). The sterileanthers display uncontrolled production of sporopollenin and lackthe sudden change ('blocking') in the heavy metal stainabilitywhich appears in the prolamellar vesicles of tapetal plastids andother structures of the tapetum (1). The coupled oxidation ofcarotenoids by LOX has been found to be associated with one(17), or two (26) isoenzymes in soybean, and the cooxidationactivity has been able to be enhanced with sulfhydryl groupmodifiers (29).Using SDS-PAGE methods, I concluded that the sterile anthers

have abnormal proteolysis after the developmental stage, when,in fertile anthers, sporopollenin production is terminated in asso-ciation with the blocking phenomena (3). In sterile anthers, spo-ropollenin secretion continues. The proteolysis is preceded by theabsence or decreased content of two polypeptides in the sterileanthers. Soluble amino N contents were determined in anthers inorder to find if there is any abnormally low or high amino N poolat the senescence.

MATERIALS AND METHODS

Plant Material. Barley (Hordeum vulgare L.) plants were grownin a greenhouse on Finnpeat B2 at a density of one plant per 49cm2. Supplementary light was provided using Airam 500Wblended-light lamps and Floralux fluorescent tubes. The plantswere not subjected to any noticeable nutritional stress. The count-able nuclear isogeny with cv. Adorra is presented in the Tablesand legends of Figures. Leaves or spikes of suitable age werecollected in Minigrip bags, kept in ice in the dark, and preparedfor assay for 1 to 3 h in random order.LOX Activity in Leaf Tissue. The plants were at early to late

anthesis. The laminas of the flag and second leaf of the top of twoplants were pooled and homogenized with a mortar and pestlewith some purified quartz sand. The working temperature was+0 to +6°C. The homogenization buffer was a modification ofthat of Grossman et al. (18): Na-phosphate, 200 mm (pH 6.8),containing 0.5% (v/v) of Triton X-100 (Koch-Light). Four ml wasused for 1 g fresh weight. The supernatant after centrifugation at20,000g for 10 min was used for the enzyme assay with or withoutpurification through Sephadex G-25M (PD-10 columns). Theelution buffer was as above but without Triton. The coupledoxidation procedure was carried out at pH 6.5 according toGrossman and Zakut (20), using a Beckman DB recording spec-trophotometer. Linoleic acid (approximately 99%) and fl-carotene(type I) were purchased from Sigma. The reaction was essentiallylinear during the 2 min recorded when 50 ,ul of the enzymesolution was made up to 1 ml. The lowering of fl-carotene con-centration by 1 nmnol under these conditions was determined tocause a decrease of 0.057 in the absorbance. This value was usedas the basis of the enzyme unit. Bleaching without added linoleatewas slow, and taken as zero in the calculations.LOX Activity in Anthers. The extraction was carried out in an

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CYTOPLASMIC MALE STERILE BARLEY

Table I. Lipoxygenase Activity in Pooled Flag Leafand Second Leaf Laminas at the Anthesis StageMeans of three determinations, each containing two plants. The relative values are presented in parentheses.

Figures followed by the same letter are not significant, otherwise significant at P < 0.05 according to SNK test.

Coupled Oxidation of ,8-CaroteneNu-clear Factor

Cyto- Restorer Anther Iso-Purified for

plasm Genest Pheno- geny with thetype with Sephadex Sepha-

cv. G-25 dex-Adorra treat-

ment

nmollmin nmollmin 102 X 102 X% ngfresh wt nmg Chli nmol/min nmol/min

. mgprotein * mg proteinAdorra +/+ Fertile 100 84.9 (100) a 229 (100) a 251 (100) a 489 (100) a 1.96 amsml Rfmla/+ Fertile 98 93.9 (ll1) a 245 (107) a 255 (102) a 487 (99) a 1.91 amsml +/+ Sterile 98 49.4 (58) 136 (59) 148 (59) 391 (80) a 2.76

F 12.917b 8.386c 5.788c 2.315 6.318Ca Plus sign (+) refers to the recessive allele of cv. Adorra.b Significant at P < 0.01.c Significant at P < 0.05.

Table II. Lipoxygenase Activity in Anther HomogenatesMeans of six determinations, each containing 45 anthers. Figures fol-

lowed by the same letter are not significant, otherwise significant at P <0.05 according to SNK test. The anther stages correspond to early and latestage 3.

Nu- Stage, Days beforeAnther clear AnthesisCyto- Restorer Peo sgn

plasm Genes' ~Pheno- Isogenyplasm Genesatype with cv. 54 2-1

Adorra

% 102 x nmol /3-caro-tene/min * anther

Adorra +/+ Fertile 96 101.1 a 109.9 aAdorra Rfmla/Rfmla Fertile 96 108.0 a 105.5 amsml Rfmla/+ Fertile 96 104.1 a 102.2 amsml +/+ Sterile 96 98.2 a 73.3

F 2.601 30.812ba Plus sign (+) refers to the recessive allele of cv. Adorra.bSignificant at P < 0.001.

ice bath. Forty-five anthers at approximately the same stage (5-4or 2-1 days before the anthesis) were homogenized in 25 ,ul Tritoncontaining (0.5%, v/v) sodium buffer (200 mm [pH 6.8]) in a cone-tipped Eppendorf tube as described previously (3), and the volumewas increased to 400 ,ul with buffer. After centrifugation at 8,000gfor 10 min, 35 IlI of the supernatant was assayed for LOX activityas above.

Electrophoresis of LOX. Eighty anthers were homogenized asabove, in 25 ,ul of the Triton buffer. After homogenization, 75 ,ulof the buffer containing 6% (v/v) of glycerol was added, and thesuspension was clarified by centrifugation at 8,000g for 7 min.Forty-five p1 of the supernatant was applied to 1-cm wide wells inslab gel. The rest was stored at -21°C for SDS-PAGE. Thepolyacrylamide gels consisted of a 5% (w/v) stacking gel (Tris-HCl, 125 mm [pH 6.81) and a 7% separation gel (Tris-HCl, 375mM [pH 9.0]). The tank buffer was Tris-glycine (pH 8.3) (11). Thegels were prepared and run as described previously (3). The gelswere stained for LOX localization according to deLumen andKazeniac (23), with the exception that linoleic acid was dispersed

with ethanol containing Tween 80 in McIlvaine citrate-phosphatebuffer (pH 6.5). Ethanol is not inhibitory at low concentrations(20). The final concentrations were linoleic acid 2 mM, ethanol1%, Tween 80 0.01%, KCN 0 or 1 mm. The staining with3,3'dimethoxybezidine HCl (Sigma) took 2 to 3 h, during whichtwo freshly prepared lots of dye in distilled H20 were used. Thereaction zone was usually cut off the gel, and stored at -21 °C forSDS-PAGE.SDS-PAGE. The frozen gel pieces carrying the LOX were

crushed in 55 yd of the sample buffer containing SDS (3). Theremaining homogenate of the anthers was also suspended in 40,ul of the sample buffer and 0.475 jig of extra SDS. The sampleswere boiled and run on SDS polyacrylamide gels, and stainedwith Brilliant Blue R as described previously (3). The mol wt wereestimated from the curvilinear plot of the mobilities of standards.The mol wt of the standards were taken from (31) or from thevalue given by the supplier: f8-galactosidase (130,000); rabbitmuscle phosphorylase a (94,000); BSA (68,000); ovalbumin(43,000); trypsinogen (24,000); Cyt c (12,400).

Determination of Chi, protein and soluble amino N. Chl wasdetermined in 80% acetone (6). Proteins after Sephadex G-25Mpurification were determined spectrophotometrically in 40 mmphosphate buffer at pH 7.2 according to Waddell (25, 30) usingBSA as the standard. The soluble amino N in the supernatantswas determined using the ninhydrin method (33) after precipita-tion with TCA with leucine (Fluka, puriss) as the standard.

RESULTS

LOX Activity. The activities in the pooled, two highest laminasare shown in Table I. The restored msml plants (heterozygous forthe restorer (Rfmla/+) had a similar activity on both a freshweight, Chl, and protein basis than the almost isogenic normalbarley cv. Adorra. The activity of the unrestored male sterileplants was the lowest. Thus, the homogenates of the fertile formsirrespective of the cytoplasm appear to have a higher activity thanthe male sterile at this stage.The removal of potential inhibitors of low mol wt with Sepha-

dex raised the activity of male fertile genotypes by factors 1.91 or1.96, and that of the male sterile genotype by a factor of 2.76. Thissuggests that the concentration of inhibitors in the extract of themale sterile plant is significantly higher, though necessarily not

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Plant Physiol. Vol. 69, 1982

FIG. 1. PAGE and SDS-PAGE slab gels. The mol wt standards are presented on the left margin in kilodaltons. The small numbers indicate thedifferent zones: (1), 91,200; (2), 70,800; (3), 67,900; (4), 57,500 dalton. The plants are given in capital letters at the bottom: (A), Adorra-like with Adorracytoplasm, and without restorer genes, +/+; (S), Adorra-like with msml cytoplasm, and without restorer genes, +/+; (F), Adorra-like with msmlcytoplasm, heterozygous for restorer gene, Rfmla/+. Each plant has a calculated nuclear isogeny of 96 to 100% with cv. Adorra.-(a, b, c, d, and e), Theupper gel (without SDS) shows the places from where the lipid-LOX zones were cut and run on the SDS gel below. Material of a, b, and c is from stage1 anthers during which sporopollenin is deposited on exines (3), and that of d and e from stage 2 anthers when the sporopollenin depositing isterminating (3). (f and g), The original anther homogenates run on the same SDS gel as tracks a through e;f is homogenate of a without the 91,200dalton polypeptide, and g is homogenate of d carrying the 91,200 polypeptide indicated with (1). (h and i), Anthers of stage 2 prepared with method 1which removes small molecules (3). Material of h is from sterile anthers indicating the absence of the 91,200 polypeptide, and that of i is from fertileanthers displaying zone (1). (j, k and 1), Chloroplast membrane proteins prepared from leaf tissue and run on SDS gel as described (3). The three minorzones indicated have the same relative mobilities as the three polypeptides (2), (3), and (4) in comparison with standards.

high enough to explain the difference in activities.Two stages of anthers were studied for LOX activity. The

activity in developing anthers seems to be considerably higherthan that in the fully-expanded leaf tissue. The activity is notsignificantly lower in sterile than in fertile anthers 4 or 5 daysbefore anthesis on a single anther basis. One or 2 days beforeanthesis, the sterile anthers have gradually collapsed, and theirLOX activity is significantly lower than that in the fertile anthers,whose activity is approximately the same per anther as some 3days before (Table II).

Electrophoresis of Anther LOX. Even before staining of the

gels with 3,3'-dimethoxybenzidine HCI, the apparently reactedzones could be localized by their transparent appearance. Thetransparent zones had evidently dissolved the linoleate, while therest of the gel was covered with an opaque emulsion. The un-stained zones were often greenish and obviously contained plas-tidic pigment in addition to lipid.The mol wt estimates of the high end on SDS-PAGE changed

from the previous ones when a linear equation was applied witha less extending set of standards (3). The polypeptide previouslycalled 70 kD, was now determined to have a mol wt of 91,200using the present standards and curvilinear estimation.

270 AHOKAS

4m,; --Wximom*-:001151FAM"

Aowmw

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CYTOPLASMIC MALE STERILE BARLEY

ab cd e f

A A A S F DFIG. 2. Lipid-LOX zones (cut from the gel) showing the increase in

mobility with age of the anthers. The print is cut at the beginning of theseparation gel and at the front. The plants are given in capital letters at thebottom: (D), Adorra-like with Adorra cytoplasm, homozygous for restorergene, Rfmla/Rfmla; the other keys are presented in the legend to Figure1. Each plant has a calculated nuclear isogeny of 96 to 100%o with cv.Adorra. Stage of anthers: (c), stage 1; (b and d), stage 2; (a, e and f),stage 3.

Table III. Soluble Amino Nitrogen Content in Anther HomogenatesMeans of six determinations, each containing 45 anthers. Figures fol-

lowed by the same letter are not significant, otherwise significant at P <0.05 according to SNK test. The anther stages correspond to early and latestage 3.

Nu- Stage, Days before

Cyto- Restorer Anther clear Anthesis

plasm Genes' Pheno- Isogenytype with cv. 5-4 2-1

Adorranmol amino N/anther

Adorra +/+ Fertile 96 23.7 a 45.3 aAdorra Rfmla/Rfmla Fertile 96 24.3 a 49.6 amsml Rfmla/+ Fertile 96 22.2 a 50.5 amsml +/+ Sterile 96 13.7 14.0

F 29.255b 79.657ba Plus sign (+) refers to the recessive allele of cv. Adorra.b Significant at P < 0.001.

During stage 1 (3), when sporopollenin is depositing on themicrospore exines, the LOX activity zone displays little mobilityon the separation gels- 1 to 5% of the mobility of the front (Figs.1 and 2). An SDS-PAGE of the extract of such a zone reveals onepolypeptide of 57,500 dalton, another of 67,900, and usually athird fainter zone of 70,800 dalton is visible. The different geno-

types studied do not appear to differ from each other in thesepolypeptides at this stage (Fig. 1, a-c).

At stage 2 (3), during which the production of sporopolleninterminates in the fertile anthers, the mobility of the LOX zonesincreases in all the genotypes to about double that at stage 1 (Figs.1 and 2). The SDS-PAGE of the active zone reveals an additionalpolypeptide associated with the LOX zone in both the male fertilegenotypes. This polypeptide is missing from the LOX zones of themsml sterile anthers. It has an apparent mol wt of 91,200 dalton.The coelectrophoresis with the original anther homogenate con-taining SDS, this zone had the same relative position to the otherpolypeptides and the same movement as the late polypeptidepreviously called 70 kD (3). This polypeptide was found only in

the homogenates of fertile anthers from stage 2 on, and wasabsent, or at the best, very faint in those of sterile anthers (Fig. 1,d-i).At stage 3, the mobility of the LOX zones appeared to increase

further (Fig. 2). The increase in mobility seems to be gradual fromstage 1 through the later stages. On SDS-PAGE gels, the stage 3LOX zones of fertile anthers continuously carried the three poly-peptides of apparent mol wt of 57,500, 67,900, and 91,200. Thefaint zone of 70,800 is obscure at stage 2 and 3.The staining of the LOX zone was not inhibited by KCN (1

mM) in the substrate, suggesting that the peroxidation is not dueto heme proteins. Gel pieces cut from the LOX zones had a slow,B-carotene bleaching activity, while a random gel piece from thearea of 50%o mobility did not bleach ,B-carotene at all. Theseobservations show that the zones concerned were probably carry-ing the LOX enzyme or enzymes.

Soluble Amino N. The soluble amino N content of the antherhomogenates was found to be low in the sterile anthers duringboth the stages studied, whereas the fertile anthers have a signif-icantly higher content which seems to increase towards the mat-uration of the anther (Table III).

DISCUSSION

A major part of the lower LOX activity in the leaf tissue ofmale sterile plants can be ascribed to endogenous inhibitors. Inpea foliage, LOX activity could be lowered by exogenous appli-cations with cytokinins, BA, or kinetin (19, 22). They suggestedthat the increase in LOX activity is associated with senescence inpea foliage (19). In vitro, the cooxidation activity ofsoybean LOX-1 has been increased with artificial modification of the sulfhydrylgroups (29). Recently, especially the lamellar LOX activity ofwheat plastid LOX has been shown to be inhibited with exogenousphenolics (15). Using several criteria, Douillard (12) has found adecrease in LOX activity in primary wheat leaf with aging. Thus,if the stem leaves of barley resemble the case with wheat primaryleaf, the male sterile msml plant leaf tissue may be regarded asphysiologically older than that of the fertile genotypes at theanthesis stage.The physiological functions of LOX are poorly understood (19,

20). The high LOX activity in the anthers compared to that of leaftissue was perhaps not caused artificially, and this therefore sug-gests that LOX(s) are important in anthers. These enzymes maybe associated with the production of oxidized carotenoids to serveas precursors of sporopollenin. The LOX activity in sterile anthersappears almost at the normal level still 4 to 5 days before anthesis,thus possibly not being a limiting factor in their late occurringsporopollenin production. Since the sporopollenin secretion inbarley occurs from the tapetum (1), at least part of the LOXactivity should be localized in the tapetum, especially in tapetalplastids, since LOX is detectable in plastids (12, 14, 15). It hasbeen suggested that some LOX isoenzymes are specialized intocarotenoid cooxidizers (17, 26).The plastidic pigments-protein complex concepts seem to have

evolved alongside the development of the methods (10, 24). TheLOX activity zone of the anthers may also be a lipid-proteincomplex whose composition may have been affected by the pres-ence of Triton. The polypeptides of 57,500, 67,900, and 70,800dalton appearing in this complex probably have in chloroplastmembranes equivalent polypeptides with the same relative mobil-ities (Fig. 1, j-l). All or at least two of the chloroplast polypeptidesevidently exist in the chlorophyll-protein complex I (21). Thisconcept of equivalence is strengthened by the knowledge thatLOX is partly bound to chloroplastidic lamellas in wheat (14).

If the polypeptide of 91,200 dalton appears in substantialamounts of tapetum, this protein may play a key role in theblocking phenomena (1). There is no blocking in sterile anthers,but it starts in fertile anthers at the stage when the 91,200 poly-

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Plant Physiol. Vol. 69, 1982

peptide is detectable. The blocking in its cytological stainingproperties resembles the formation of trilinolein from exogenouslinolenic acid in fibroblasts (27). Since substantial amounts of the91,200 polypeptide is left in the anther homogenates (Fig. lg), thepresence of this protein in the LOX zone may be due to its bindingto its insoluble substrate in the zone.While most LOX are reported to have mol wt of about 100,000,

for pea LOX a mol wt of 67,000, 72,000, or 78,000, and forbroadbean LOX one of 88,000 have been reported (5, 7, 16).Though two isoenzymes have been found in germinating barley(32), the mol wt are unknown.The pseudoisoenzymic movement of LOX zones on gels has

likely its origin in the change of the lipid component with time.Douillard and Bergeron (13) have found a decrease in the densityof plastidic lamellas carrying LOX on aging of the leaf tissue.

There is a depletion of the amino N pool of the sterile anthers,which appears to be normalized in the restored derivatives. Thelowering of its sink capacity, or otherwise different distribution ofN in the plant body, probably has drastic consequences for theanther, decreasing its ability to synthesize late proteins (e.g. thatof the 91,200 dalton) and influencing the stability of the proteinsin general. The evident N starvation may be an important reasonin the chain of events leading to anther sterility, which is reinforcedby the wasteful production of sporopollenin in msml sterile an-thers (1). Sinks are regulated by hormones, especially by cytoki-nins, which may be concerned in fertility restoration (3).

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