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 con- centration 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.