Fertile revertants from S-type male-sterile maize grown in vitro.

Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3-4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13-20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.

Mitochondrial DNA duplication/deletion events and polymorphism of the C group of male sterile maize cytoplasms.

Five accessions of members of the C group of male sterile maize cytoplasms (BB, C, ES, PR, and RB) in two nuclear backgrounds (A619 and A632) were examined to elucidate the nature of mitochondrial genome diversity within a related group of cytoplasms. Cosmid and plasmid clones carrying single copy and recombinationally active sequences from N and S cytoplasms of maize were used as probes. Although restriction patterns are quite similar, each of the five could be discriminated by evidence of sequence duplication and recombination, deletion of recombinationally active sequences of N, normal cytoplasm, population of mini-circular DNAs, and by restriction patterns. Each member of the group carried a 1,913 bp minicircular mtDNA, while all entries but RB carried a 1,445 bp minicircular mtDNA. Members of the C group clearly are not molecularly identical; evolution of the group included principal genome reorganization involving sequence duplication/deletion events, apparently independent of the cms trait.

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.).

Differences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.

A new source of cytoplasmic male sterility in maize induced by the nuclear gene, iojap.

Cytoplasmic male sterility (cms) was found in plants derived from the F2 progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.

Genetics of fertility restoration in the C-group of cytoplasmic male sterility in maize.

The genetics of fertility restoration of cms-C group cytoplasm of maize was studied using crosses involving stable maintainer lines and lines that restored full pollen fertility. Pollen fertility in the sources of cms-C sterile cytoplasms studied was restored by a single dominant restorer (Rf4) gene. The fertility restoration was sporophytic. Allelism tests among five restorer lines showed that they all apparently carried the same alleles (Rf4 Rf4). Similar tests also demonstrated that seven nonrestoring maintainer lines had apparently the same genotype (rf4 rf4), although a partial "late break" of fertility was observed at low levels in some maintainer crosses. Comparative studies among different cms-C sources (C, Bb, ES, PR and RB) indicated that similar inheritance of fertility restoration was involved. The data indicated that a single, dominant Rf gene is involved in the restoration of several C-group cytoplasms, at least in the lines studied here. This is the first single-gene, sporophytic restorer system described in maize to date.

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts.

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.

Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria.

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD(+)-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD(+). Added NAD(+) partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD(+)-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD(+) and other coenzymes.

Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts.

High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 mug dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 mug of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action.

Evaluation of a Chemical Method for Assay of Helminthosporium maydis Race T Toxin.

A chemical test reported by Karr et al. (Plant Physiol. 55:727) to assay for host-specific toxin produced by Helminthosporium maydis race T was evaluated. Preparations from culture filtrates of both race T isolates, containing host-specific toxin, and similar preparations from race O isolates, containing no detectable host-specific toxin, gave positive reactions in the chemical assay. Also, preparations containing active or inactive toxin gave equal responses in this test. The procedure does not provide a reliable method for assaying H. maydis race T toxin.

Ultrastructural effects of Helminthosporium maydis race T toxin on mitochondria of corn roots and protoplasts.

Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.

Biochemical and cytological relationships in C4 plants.

C4 plants can be divided into three groups based on differences in activities of three decarboxylating enzymes: NADP-malic enzyme, NAD-malic enzyme, and phosphopyruvate carboxykinase.In the Gramineae the three C4 groups are distinguished by anatomical and ultrastructural characteristics of bundle-sheath chloroplasts. NADP-malic enzyme species lack well-developed grana in bundle-sheath chloroplasts (grana reduced) and the bundle-sheath chloroplasts are in the centrifugal position. NAD-malic enzyme species have bundle-sheath chloroplasts in the centripetal position and contain grana. Phosphopyruvate carboxykinase species have bundle-sheath chloroplasts in the centrifugal position and they contain grana. NADP-malic enzyme species of the Gramineae have only been found in the subfamilies Aristidoideae and Panicoideae. With the exception of the genera Panicum, and Urochloa, NAD-malic enzyme species and phosphopyruvate carboxykinase species have only been found in the subfamily Eragrostoideae. C4 species of the genus Panicum are found among all three of the C4 groups.The dicotyledonous C4 species examined fall into two groups: those having high NADP-malic enzyme and those having high NAD-malic enzyme. No phosphopyruvate carboxykinase C4 species have been found among the dicotyledons. The NADP-malic enzyme C4 species of the dicotyledons like NADP-malic enzyme species of the Gramineae have bundle-sheath chloroplasts with reduced grana but in contrast to NADP-malic enzyme species of the Gramineae the bundle-sheath chloroplasts are in the centripetal position. The NAD-malic enzyme species of the dicotyledons like the NAD-malic enzyme species of the Gramineae have bundlesheath chloroplasts in the centripetal position with well developed grana.The results are discussed in terms of evolutionary and functional diversification of C4 plants.

Peripheral reticulum in chloroplasts of plants differing in CO2 fixation pathways and photorespiration.

The development of peripheral reticulum (PR) in chloroplasts varies in C3 and C4 plants. In general, PR is more extensive in C4 plants, but PR is also seen in the chloroplasts of some C3 plants. Within some C4 plants, PR is seen in the bundle sheath cells which predominantly use the C3 pathway. Thus, PR is not associated directly with the presence of the C4 pathway on a cellular basis. Its predominance in C4 plants must be related to some characteristic other than the method of CO2 fixation. Ultrastructural evidence suggests that PR is associated with the rapid transfer of substances into and out of chloroplasts and from mesophyll to bundle sheath cells.

Leaf microbodies (peroxisomes) and catalase localization in plants differing in their photosynthetic carbon pathways.

The tropical grasses sugarcane (Saccharum officinarum) and pangolagrass (Digitaria decumbens) contained fewer leaf microbodies than temperate orchardgrass (Dactylis glomerata). Leaf microbodies were seen in both the mesophyll and bundle sheath cells of tropical grasses. The fibrous elements in the microbodies of tropical grasses differed from those of the temperate grass. Catalase was predominantly localized in the microbodies of leaf cells (3,3'-diaminobenzidine method). The site of greatest catalase activity appeared to be the fibrous and/or crystalline inclusions within the microbodies. The low rates of photorespiration noted in tropical grasses do not appear to be due to the complete absence of the necessary organelles.