Cytoplasmic Male Sterility in Barley' VIII

Plant Physiol. (1982) 69, 0268-0272 0032-0889/82/69/0268/05/$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 AHOKAS Department of Genetics, University ofHelsinki, P. Rautatiekatu 13, 00100 Helsinki 10, Finland

ABSTRACT lenin in the tapetum. Sporopollenin is evidently an oxidative polymer of carotenoids or carotenoid esters (8, 9). The sterile The lipoxygenase (LOX) activity was determined in almost isogenic anthers display uncontrolled production ofsporopollenin and lack types of barley (Hordeum vulgare L.): normal cv. Adorra, cytoplasmic male the sudden change ('blocking') in the heavy metal stainability sterile (msml), and msmi barley with restored fertility, heterozygous for which appears in the prolamellar vesicles of tapetal plastids and the Rfmila restorer gene. The LOX activity was lowest in male steriles in other structures of the tapetum (1). The coupled oxidation of the leaf tissue studied at the anthesis stage. The LOX activity in developing carotenoids by LOX has been found to be associated with one anthers was higher than in leaf tissue, and decreased during degeneration (17), or two (26) isoenzymes in soybean, and the cooxidation of the sterile anthers. activity has been able to be enhanced with sulfhydryl group On polyacrylamide gel electrophoresis slabs, the LOX of anther homog- modifiers (29). enates moved in a complex which evidently carried some lipid and pigment, Using SDS-PAGE methods, I concluded that the sterile anthers too. The LOX zones showed pseudoisoenzymic movement, Le. a gradual have abnormal proteolysis after the developmental stage, when, increase in mobility dependent on the age of the anthers. On sodium in fertile anthers, sporopollenin production is terminated in asso- dodecyl sulfate-polyacrylamide gel electrophoresis slabs, the LOX zones ciation with the blocking phenomena (3). In sterile anthers, spo- contained three polypeptides. When sporopolienin production ends in the ropollenin secretion continues. The proteolysis is preceded by the fertile anthers, a fourth polypeptide (molecular weight 91,200) appears in absence or decreased content of two polypeptides in the sterile their LOX zones. This 'late' polypeptide is missing from steriles, and is anthers. Soluble amino N contents were determined in anthers in suggested as being associated with the termination of sporopollenin pro- order to find if there is any abnormally low or high amino N pool duction in the tapetum of fertiles. at the senescence. Sterile anthers were found to be almost devoid of soluble NH2-N, which supports the idea of their starvation. This starvation can reasonably be held responsible for the absence of late proteins (e.g. the 91,200 dalton MATERIALS AND METHODS polypeptide), and is reinforced by the uncontrolled production of sporopol- Plant Material. Barley (Hordeum vulgare L.) plants were grown lenin. in a greenhouse on Finnpeat B2 at a density of one plant per 49 cm2. Supplementary light was provided using Airam 500W blended-light lamps and Floralux fluorescent tubes. The plants were not subjected to any noticeable nutritional stress. The count- able nuclear isogeny with cv. Adorra is presented in the Tables and legends of Figures. Leaves or spikes of suitable age were collected in Minigrip bags, kept in ice in the dark, and prepared Cytoplasmic male sterility, or male sterile maternal 1 (msml), for assay for 1 to 3 h in random order. was found as a natural variant in an Israeli strain of the wild LOX Activity in Leaf Tissue. The plants were at early to late progenitor of barley (Hordeum vulgare ssp. spontaneum [C. Koch] anthesis. The laminas of the flag and second leaf of the top of two Thellung) (2). The original strain carries a dominant fertility plants were pooled and homogenized with a mortar and pestle restorer gene designated as Rfmla. A number of restorer genes, as with some purified quartz sand. The working temperature was well as partial restorer genotypes, have been found in wild barley +0 to +6°C. The homogenization buffer was a modification of strains from Israel (4, and unpublished). These additional restorer that of Grossman et al. (18): Na-phosphate, 200 mm (pH 6.8), genes appear in male fertile cytoplasms. This finding and pecu- containing 0.5% (v/v) of Triton X-100 (Koch-Light). Four ml was liarities in their geographic distribution suggest that the restorer used for 1 g fresh weight. The supernatant after centrifugation at genes are responsible for special ecophysiological adaptations. 20,000g for 10 min was used for the enzyme assay with or without Another, different type of maternal male sterility (msm2) was purification through Sephadex G-25M (PD-10 columns). The recently found by the author (in preparation). elution buffer was as above but without Triton. The coupled LOX2 activity in leaf and anther tissue in various, near-isogenic oxidation procedure was carried out at pH 6.5 according to derivatives of msml and normal barley is described in this paper. Grossman and Zakut (20), using a Beckman DB recording spec- The interest in LOX activity takes two forms. First, LOX is located trophotometer. Linoleic acid (approximately 99%) and fl-carotene either entirely in plastids (12, 13), or else partly in mitochondria (type I) were purchased from Sigma. The reaction was essentially (28) and may, therefore, be associated with maternal inheritance. linear during the 2 min recorded when 50 ,ul of the enzyme Second, LOX may be associated with the production of sporopol- solution was made up to 1 ml. The lowering of fl-carotene concentration by 1 nmnol under these conditions was determined to 'The work was done under the auspices of the Research Council for cause a decrease of 0.057 in the absorbance. This value was used Agriculture and Forestry of the Academy of Finland. as the basis ofthe enzyme unit. Bleaching without added linoleate 2Abbreviations: LOX, lipoxygenase, linoleate: oxygen, oxidoreductase was slow, and taken as zero in the calculations. EC 1. 13.11.12; PAGE, polyacrylamide gel electrophoresis. LOX Activity in Anthers. The extraction was carried out in an 268 CYTOPLASMIC MALE STERILE BARLEY 269 Table I. Lipoxygenase Activity in Pooled Flag Leafand Second Leaf Laminas at the Anthesis Stage Means 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-Carotene Nu- clear Factor Anther Iso- Cyto- Restorer Purified for plasm Genest Pheno- geny with the type 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 protein Adorra +/+ Fertile 100 84.9 (100) a 229 (100) a 251 (100) a 489 (100) a 1.96 a msml Rfmla/+ Fertile 98 93.9 (ll1) a 245 (107) a 255 (102) a 487 (99) a 1.91 a msml +/+ Sterile 98 49.4 (58) 136 (59) 148 (59) 391 (80) a 2.76

F 12.917b 8.386c 5.788c 2.315 6.318C a 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 Homogenates with ethanol containing Tween 80 in McIlvaine citrate-phosphate Means of six determinations, each containing 45 anthers. Figures fol- buffer (pH 6.5). Ethanol is not inhibitory at low concentrations lowed by the same letter are not significant, otherwise significant at P < (20). The final concentrations were linoleic acid 2 mM, ethanol 0.05 according to SNK test. The anther stages correspond to early and late 1%, Tween 80 0.01%, KCN 0 or 1 mm. The staining with stage 3. 3,3'dimethoxybezidine HCl (Sigma) took 2 to 3 h, during which two freshly prepared lots of dye in distilled H20 were used. The Nu- Stage, Days before reaction zone was usually cut off the gel, and stored at -21 °C for Cyto- Restorer AntherPeo sgnclear Anthesis ~Pheno- SDS-PAGE. plasmplasm Genes'Genesa Isogeny SDS-PAGE. The frozen gel pieces carrying the LOX were type with cv. 54 2-1 Adorra crushed in 55 yd of the sample buffer containing SDS (3). The remaining homogenate of the anthers was also suspended in 40 % 102 x nmol /3-caro- ,ul of the sample buffer and 0.475 jig of extra SDS. The samples tene/min * anther were boiled and run on SDS polyacrylamide gels, and stained Adorra +/+ Fertile 96 101.1 a 109.9 a with Brilliant Blue R as described previously (3). The mol wt were Adorra Rfmla/Rfmla Fertile 96 108.0 a 105.5 a estimated from the curvilinear plot of the mobilities of standards. msml Rfmla/+ Fertile 96 104.1 a 102.2 a The mol wt of the standards were taken from (31) or from the msml +/+ Sterile 96 98.2 a 73.3 value given by the supplier: f8-galactosidase (130,000); rabbit muscle phosphorylase a (94,000); BSA (68,000); ovalbumin F 2.601 30.812b (43,000); trypsinogen (24,000); Cyt c (12,400). a Determination of Chi, protein and soluble amino N. Chl was Plus sign (+) refers to the recessive allele of cv. Adorra. determined in 80% acetone (6). Proteins after Sephadex G-25M bSignificant at P < 0.001. purification were determined spectrophotometrically in 40 mm phosphate buffer at pH 7.2 according to Waddell (25, 30) using ice bath. Forty-five anthers at approximately the same stage (5-4 BSA as the standard. The soluble amino N in the supernatants or 2-1 days before the anthesis) were homogenized in 25 ,ul Triton was determined using the ninhydrin method (33) after precipita- containing (0.5%, v/v) sodium buffer (200 mm [pH 6.8]) in a cone- tion with TCA with leucine (Fluka, puriss) as the standard. tipped Eppendorftube as described previously (3), and the volume was increased to 400 ,ul with buffer. After centrifugation at 8,000g RESULTS for 10 min, 35 IlI of the supernatant was assayed for LOX activity as above. LOX Activity. The activities in the pooled, two highest laminas Electrophoresis of LOX. Eighty anthers were homogenized as are shown in Table I. The restored msml plants (heterozygous for above, in 25 ,ul of the Triton buffer. After homogenization, 75 ,ul the restorer (Rfmla/+) had a similar activity on both a fresh of the buffer containing 6% (v/v) of glycerol was added, and the weight, Chl, and protein basis than the almost isogenic normal suspension was clarified by centrifugation at 8,000g for 7 min. barley cv. Adorra. The activity of the unrestored male sterile Forty-five p1 of the supernatant was applied to 1-cm wide wells in plants was the lowest. Thus, the homogenates of the fertile forms slab gel. The rest was stored at -21°C for SDS-PAGE. The irrespective of the cytoplasm appear to have a higher activity than polyacrylamide gels consisted of a 5% (w/v) stacking gel (Tris- the male sterile at this stage. HCl, 125 mm [pH 6.81) and a 7% separation gel (Tris-HCl, 375 The removal of potential inhibitors of low mol wt with Sepha- mM [pH 9.0]). The tank buffer was Tris-glycine (pH 8.3) (11). The dex raised the activity of male fertile genotypes by factors 1.91 or gels were prepared and run as described previously (3). The gels 1.96, and that of the male sterile genotype by a factor of 2.76. This were stained for LOX localization according to deLumen and suggests that the concentration of inhibitors in the extract of the Kazeniac (23), with the exception that linoleic acid was dispersed male sterile plant is significantly higher, though necessarily not 270 AHOKAS Plant Physiol. Vol. 69, 1982

Aowmw

4m,; 001151FAM" --Wximom*-: aw".., i.

FIG. 1. PAGE and SDS-PAGE slab gels. The mol wt standards are presented on the left margin in kilodaltons. The small numbers indicate the different 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 Adorra cytoplasm, and without restorer genes, +/+; (S), Adorra-like with msml cytoplasm, and without restorer genes, +/+; (F), Adorra-like with msml cytoplasm, 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), The upper 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 stage 1 anthers during which sporopollenin is deposited on exines (3), and that of d and e from stage 2 anthers when the sporopollenin depositing is terminating (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 91,200 dalton 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 1 which 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 fertile anthers displaying zone (1). (j, k and 1), Chloroplast membrane proteins prepared from leaf tissue and run on SDS gel as described (3). The three minor zones 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. gels with 3,3'-dimethoxybenzidine HCI, the apparently reacted Two stages of anthers were studied for LOX activity. The zones could be localized by their transparent appearance. The activity in developing anthers seems to be considerably higher transparent zones had evidently dissolved the linoleate, while the than that in the fully-expanded leaf tissue. The activity is not rest of the gel was covered with an opaque emulsion. The un- significantly lower in sterile than in fertile anthers 4 or 5 days stained zones were often greenish and obviously contained plas- before anthesis on a single anther basis. One or 2 days before tidic pigment in addition to lipid. anthesis, the sterile anthers have gradually collapsed, and their The mol wt estimates of the high end on SDS-PAGE changed LOX activity is significantly lower than that in the fertile anthers, from the previous ones when a linear equation was applied with whose activity is approximately the same per anther as some 3 a less extending set of standards (3). The polypeptide previously days before (Table II). called 70 kD, was now determined to have a mol wt of 91,200 Electrophoresis of Anther LOX. Even before staining of the using the present standards and curvilinear estimation. CYTOPLASMIC MALE STERILE BARLEY 271 the homogenates of fertile anthers from stage 2 on, and was ab cd e f absent, 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 from stage 1 through the later stages. On SDS-PAGE gels, the stage 3 LOX zones of fertile anthers continuously carried the three polypeptides of apparent mol wt of 57,500, 67,900, and 91,200. The faint 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 due to heme proteins. Gel pieces cut from the LOX zones had a slow ,B-carotene bleaching activity, while a random gel piece from the area of 50%o mobility did not bleach ,B-carotene at all. These observations show that the zones concerned were probably carrying the LOX enzyme or enzymes. Soluble Amino N. The soluble amino N content of the anther A A A S F D homogenates was found to be low in the sterile anthers during FIG. 2. Lipid-LOX zones (cut from the gel) showing the increase in both the stages studied, whereas the fertile anthers have a signif- mobility with age of the anthers. The print is cut at the beginning of the icantly higher content which seems to increase towards the mat- separation gel and at the front. The plants are given in capital letters at the uration of the anther (Table III). bottom: (D), Adorra-like with Adorra cytoplasm, homozygous for restorer gene, Rfmla/Rfmla; the other keys are presented in the legend to Figure DISCUSSION 1. 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), A major part of the lower LOX activity in the leaf tissue of stage 3. male sterile plants can be ascribed to endogenous inhibitors. In pea foliage, LOX activity could be lowered by exogenous appli- Table III. Soluble Amino Nitrogen Content in Anther Homogenates cations with cytokinins, BA, or kinetin (19, 22). They suggested that the increase in LOX activity is associated with senescence in Means of six determinations, each containing 45 anthers. Figures fol- pea foliage (19). In vitro, the cooxidation activity ofsoybean LOX- lowed by the same letter are not significant, otherwise significant at P < 1 has been increased with artificial modification of the sulfhydryl 0.05 according to SNK test. The anther stages correspond to early and late groups (29). Recently, especially the lamellar LOX activity of stage 3. wheat plastid LOX has been shown to be inhibited with exogenous Nu- Stage, Days before phenolics (15). Using several criteria, Douillard (12) has found a Cyto- Restorer Anther clear Anthesis decrease in LOX activity in primary wheat leaf with aging. Thus, plasm Genes' Pheno- Isogeny if the stem leaves of barley resemble the case with wheat primary type with cv. 5-4 2-1 leaf, the male sterile msml plant leaf tissue may be regarded as Adorra physiologically older than that of the fertile genotypes at the nmol amino N/anther anthesis stage. The physiological functions of LOX are poorly understood (19, Adorra +/+ Fertile 96 23.7 a 45.3 a 20). The high LOX in Adorra Fertile 96 24.3 a activity the anthers compared to that of leaf Rfmla/Rfmla 49.6 a tissue was perhaps not caused artificially, and this therefore sug- msml Rfmla/+ Fertile 96 22.2 a 50.5 a gests that LOX(s) are important in anthers. These enzymes may msml +/+ Sterile 96 13.7 14.0 be associated with the production of oxidized carotenoids to serve as precursors of sporopollenin. The LOX activity in sterile anthers F 29.255b 79.657b appears almost at the normal level still 4 to 5 days before anthesis, a Plus sign (+) refers to the recessive allele of cv. Adorra. thus possibly not being a limiting factor in their late occurring b Significant at P < 0.001. sporopollenin production. Since the sporopollenin secretion in barley occurs from the tapetum (1), at least part of the LOX During stage 1 (3), when sporopollenin is depositing on the activity should be localized in the tapetum, especially in tapetal microspore exines, the LOX activity zone displays little mobility plastids, since LOX is detectable in plastids (12, 14, 15). It has on the separation gels- 1 to 5% of the mobility of the front (Figs. been suggested that some LOX isoenzymes are specialized into 1 and 2). An SDS-PAGE of the extract of such a zone reveals one carotenoid cooxidizers (17, 26). polypeptide of 57,500 dalton, another of 67,900, and usually a The plastidic pigments-protein complex concepts seem to have third fainter zone of 70,800 dalton is visible. The different geno- evolved alongside the development of the methods (10, 24). The types studied do not appear to differ from each other in these LOX activity zone of the anthers may also be a lipid-protein polypeptides at this stage (Fig. 1, a-c). complex whose composition may have been affected by the pres- At stage 2 (3), during which the production of sporopollenin ence of Triton. The polypeptides of 57,500, 67,900, and 70,800 terminates in the fertile anthers, the mobility of the LOX zones dalton appearing in this complex probably have in chloroplast increases in all the genotypes to about double that at stage 1 (Figs. membranes equivalent polypeptides with the same relative mobil- 1 and 2). The SDS-PAGE of the active zone reveals an additional ities (Fig. 1, j-l). All or at least two of the chloroplast polypeptides polypeptide associated with the LOX zone in both the male fertile evidently exist in the chlorophyll-protein complex I (21). This genotypes. This polypeptide is missing from the LOX zones of the concept of equivalence is strengthened by the knowledge that msml sterile anthers. It has an apparent mol wt of 91,200 dalton. LOX is partly bound to chloroplastidic lamellas in wheat (14). The coelectrophoresis with the original anther homogenate con- If the polypeptide of 91,200 dalton appears in substantial taining SDS, this zone had the same relative position to the other amounts of tapetum, this protein may play a key role in the polypeptides and the same movement as the late polypeptide blocking phenomena (1). There is no blocking in sterile anthers, previously called 70 kD (3). This polypeptide was found only in but it starts in fertile anthers at the stage when the 91,200 poly- 272 AHOKAS Plant Physiol. Vol. 69, 1982 peptide is detectable. The blocking in its cytological staining proteins. Ann NY Acad Sci 121: 404 427 12. DOUILLARD R 1980 Characterization and meaning of chloroplast lipoxygenase properties resembles the formation of trilinolein from exogenous activity. In P Mazliak, P Benveniste, C Costes, R Douce, eds, Biogenesis and linolenic acid in fibroblasts (27). Since substantial amounts of the Function of Plant Lipids. Elsevier/North-Holland, pp 121-126 91,200 polypeptide is left in the anther homogenates (Fig. lg), the 13. DOUILLARD R, E BERGERON 1979 Lipoxygenase activity distribution in young presence of this protein in the LOX zone may be due to its binding wheat chloroplast lamallae. In L-A Appelqvist, C Liljenberg, eds, Advances in the Biochemistry and Physiology of Plant Lipids. Elsevier/North-Holland, pp to its insoluble substrate in the zone. 159-164 While most LOX are reported to have mol wt of about 100,000, 14. DOUILLARD R, E BERGERON 1979 Quelquescaractristiques fonctionnelles des for pea LOX a mol wt of 67,000, 72,000, or 78,000, and for activites lipoxygenasiques foliaires soluble ou lamellaire. Consequences struc- broadbean LOX one of 88,000 have been reported (5, 7, 16). turales et fonctionnelles. Physiol Veg 17: 749-768 15. DOUILLARD R, E BERGERON 1981 Lipoxygenase activities of young wheat leaves. Though two isoenzymes have been found in germinating barley Physiol Plant 51: 335-338 (32), the mol wt are unknown. 16. ERIKSSON CE, SG SVENSSON 1970 Lipoxygenase from peas, purification and The pseudoisoenzymic movement of LOX zones on gels has properties of the enzyme. Biochim Biophys Acta 198: 449-459 likely its origin in the change of the lipid component with time. 17. GROSCH W, G LASKAWY 1979 Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme. Biochim Biophys Acta 575: 439-445 Douillard and Bergeron (13) have found a decrease in the density 18. GROSSMAN S, A BEN Aziz, P BUDOWSKI, I ASCARELLI, A GERTLER, Y BIRK, A of plastidic lamellas carrying LOX on aging of the leaf tissue. BONDI 1969 Enzymic oxidation of carotene andlinoleate by alfalfa; extraction There is a depletion of the amino N pool of the sterile anthers, and separation of active fractions. Phytochemistry 8: 2287-2293 which appears to be normalized in the restored derivatives. The 19. GROSSMAN S, Y LESHEM 1978 Lowering of endogenous lipoxygenase activity in Pisum sativum foliage by cytokinin as related to senescence. Physiol Plant 43: lowering of its sink capacity, or otherwise different distribution of 359-362 N in the plant body, probably has drastic consequences for the 20. GROSSMAN S, R ZAKUT 1979 Determination of the activity of lipoxygenase anther, decreasing its ability to synthesize late proteins (e.g. that (lipoxidase). In D Glick, ed, Methods of Biochemical Analysis, Vol 25. John of the 91,200 dalton) and influencing the stability of the proteins Wiley & Sons, New York, pp 303-329 21. HENRIQUES F, R PARK 1977 Polypeptide composition of chlorophyll-protein in general. The evident N starvation may be an important reason complexes from romaine lettuce. Plant Physiol 60: 6468 in the chain ofevents leading to anther sterility, which is reinforced 22. LESHEM Y, S GROSSMAN, A FRIMER, J ZIV 1979 Endogenous lipoxygenase control by the wasteful production of sporopollenin in msml sterile an- and lipid-associated free radical scavenging as modes of cytokinin action in thers (1). Sinks are regulated by hormones, especially by cytoki- plant senescence retardation. In L-A Appelqvist, C Liljenberg, eds, Advances in the Biochemistry and Physiology of Plant Lipids. Elsevier/North-Holland, nins, which may be concerned in fertility restoration (3). Amsterdam, pp 193-198 23. DELUMEN BO, SJ KAZENIAC 1976 Staining for lipoxygenase activity in electro- LITERATURE CITED phoretic gels. Anal Biochem 72: 428-432 24. MARKWELL JP, JP THORNBER, RT BOGGS 1979 Higher plant chloroplasts: 1. AHOKAS H 1978 Cytoplasmic male sterility in barley. II. Physiology and anther evidence that all the chlorophyll exists as chlorophyll-protein complexes. Proc cytology of msml. Hereditas 89: 7-21 Nat Acad Sci USA 76: 1233-1235 2. AHoKAs H 1979 Cytoplasmic male sterility in barley. Acta Agr Scand 29: 219- 25. MURPHY JB, MW KIES 1960 Note on spectrophotometric determination of 224 proteins in dilute solutions. Biochim Biophys Acta 45: 382-384 3. AHOKAS H 1980 Cytoplasmic male sterility in barley. V. Physiological character- 26. RAMADOSS CS, EK PISTORIUS, B AXELROD 1978 Coupled oxidation of carotene ization of the msml-Rfmla system. Physiol Plant 48: 231-238 by lipoxygenase requires two isoenzymes. Arch Biochem Biophys 190: 549- 4. AHOKAS H 1980 Cytoplasmic male sterility in barley. VII. Nuclear genes for 552 restoration. Theor Appl Genet 57: 193-202 27. SCHNEEBERGER EE, RD LYNCH, RP GEYER 1971 Formation and disappearance 5. ARENS D, W SEILMEIER, F WEBER, G KLoos, W GROSCH 1973 Purification and of triglyceride droplets in strain L fibroblasts. An electron microscopic study. properties ofa carotene co-oxidizing lipoxygenase from peas. Biochem Biophys Exp Cell Res 69: 193-206 Acta 327: 295-305 28. SIEDOW JN, ME GIRVIN 1979 The effect of propyl gallate on plant mitochondrial 6. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in electron transfer and lipoxygenase activity. Plant Physiol 63:5 Beta vulgaris. Plant Physiol 24: 1-15 29. SPAAPEN LJM, J VERHAGEN, GA VELDINK, JFG VLIEGENTHART 1980 The effect 7. BEAUX Y, R DRAPRON, J NICOLAS, JM CAILLAT 1973 La lipoxyg6nase de la feve of modification of sulfhydryl groups in soybean lipoxygenase-l. Biochim (Viciafaba L.). I. Isolement. Biochimie 55: 253-262 Biophys Acta 618: 153-162 8. BROOKS J, G SHAw 1977 Recent advances in the chemistry and geochemistry of 30. WADDELL WJ 1956 A simple ultraviolet spectrophotometric method for the pollen and spore walls. Transactions of the Bose Research Institute 40: 19-38 determination of protein. J Lab Clin Med 48: 311-314 9. BROOKS J, G SHAW 1978 Sporopollenin: a review of its chemistry, palaeochem- 31. WEBER K, M OSBORN 1969 The reliability of molecular weight determinations istry and geochemistry. Grana 17: 91-97 by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244: 4406- 10. CAMM EL, BR GREEN 1980 Fractionation of thylakoid membranes with the 4412 nonionic detergent octyl-f6-D-glucopyranoside. Resolution of chlorophyll-pro- 32. YABUUCHI S 1976 Occurrence of a new lipoxygenase isoenzyme in germinating tein complex II into two chlorophyll-protein complexes. Plant Physiol 66: 428- barley embryos. Agr Biol Chem 40: 1987-1992 432 33. YEMM EW, EC COCKING 1955 The determination ofamino-acids with ninhydrin. 11. DAvIS BJ 1964 Disc electrophoresis. II. Method and application to human serum Analyst 80: 209-213