Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.     

Analysis of chloroplast differences in leaves of rice isonuclear alloplasmic lines

The chloroplast being an important organelle of plant cells could possibly be associated with plant cytoplasmic male sterility (CMS). To better understand the correlation between (CMS) and chloroplast, we presented a comprehensive analysis based on the changes of photosynthetic parameters, chloroplasts ultrastructure, soluble sugar and starch content, the relative expression of sugar and starch metabolism genes, and chloroplast genome in rice isonuclear alloplasmic CMS lines at the flowering stage. Leaf gas exchange parameters did not affect by CMS lines (M2BS and M2A), although intercellular CO₂ concentration (C _i) was influenced in both M2BS and M2A. Ultrastructural observation results indicated that many starch granules were observed in the chloroplast of CMS lines, especially bigger size in M2BS, while few ones in M2B. Only the chloroplasts of M2A contained some additional number of lipoids compared with those of the other two lines (M2B and M2BS). Soluble sugar and starch contents in CMS lines (M2BS and M2A) were significantly higher than those in maintainer line (M2B) (p?<?0.01). The relative expression of sugar and starch metabolism genes indicated the imbalance of starch and sugar synthesis and decomposition may lead to accumulation of starch granules and demonstrated the presence of cytoplasmic effects. Moreover, chloroplast genome sequencing results showed similarity in both CMS lines, which revealed different single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) models compared with their maintainer line. Those models were located in psbD, rpoC2, rpl33, psbB, ndhA, ndhH, and intergenic regions. These findings, aligned with the possible association of CMS characteristics with cpDNA and genetically close relationship among both CMS lines, may contribute for future research.     

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome. Received: 10 September 1998 / Accepted: 24 February 1999     

Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny

Plastids and mitochondria, the DNA‐containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co‐transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild‐type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS‐92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid‐transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.     

Chloroplast DNA polymorphism in fertile and male-sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench)

Summary Restriction endonuclease patterns of chloroplast DNA (cpDNA) were consistently distinguishable between fertile and male-sterile cytoplasms of sorghum [Sorghum bicolor (L.) Moench], whereas no differences in restriction patterns of cpDNA among male-sterile (A₁) lines, including six isocytoplasmic strains, were revealed in this study. It is suggested that chloroplast DNA may contribute to the male sterility of A₁ lines used currently in hybrid sorghum production.This research was supported by a research grant from Kansas Grain Sorghum Commission, Kansas Board of Agriculture. Contribution 90-293-J from the Kansas Agricultural Experiment Station     

Restoration of male fertileNicotiana by fusion of protoplasts derived from two different cytoplasmic male-sterile cybrids

Summary Using the donor-recipient protoplast-fusion technique, we have recently constructed several alloplasmic-like lines ofNicotiana in which the original cytoplasms (or part of them) of eitherN. tabacum orN. sylvestris were replaced respectively, either byN. undulata or byN. bigelovii cytoplasms. These cybridizations resulted in two kinds of cytoplasmic male-sterile (CMS) cybrid plants:N. tabacum withN. undulata-like cytoplasm andN. sylvestris withN. bigelovii-like cytoplasm. Fusion of protoplasts, derived from the above two CMS types, by the donor-recipient technique, lead to the recovery of 21 cybrid calli. One of these regenerated a cybrid with fertile pollen but having shortened filaments and slighly tappered anthers. Self pollination of the latter cybrid resulted in a second generation progeny having almost normal filaments and anthers. Further selfings produced a third generation in which numerous plants had normal stamens and fertile pollen. Mitochondrial DNA (mtDNA) analysis of second and third generation progenies revealed a novel pattern which differed from each of the parental CMS cybrids and also from the mtDNA of normal, male-fertileNicotiana species. The results suggest that mtDNA recombination between different types of CMS can lead to restoration of male-fertility.Paper presented at the 1st Plant Molecular Biology Congress, Savannah, Georgia, Oct./Nov. 1985.     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Nuclear-cytoplasmic interactions in restoration of male fertility in the ‘9E’ and A4 CMS-inducing cytoplasms of sorghum

 The genetics of male-fertility restoration in sorghum in the “9E” and A4 CMS-inducing cytoplasms, was studied by crossing a number of fertility restorer lines of A1 cytoplasm to CMS lines [9E]T×398 and [A4]T×398 and the line [9E]Milo-10, which was obtained by backcrossing Milo-10 to [9E]T×398. It was revealed that both A4 and “9E” cytoplasms are characterized by a sporophytic mode of restoration of male fertility. Depending on the nuclear background of the male parents, fertility restoration was controlled by one or two dominant genes. Fertility-restorer genes of one of the tester lines, KVV-114, were effective in [9E]T×398 but could not restore [9E]Milo-10. A fertile line obtained from the fertile hybrid [9E]T×398/KVV-112, with “9E” cytoplasm, also failed to restore [9E]Milo-10. In a number of hybrid combinations with both A4 and “9E” cytoplasms a novel and unusual phenomenon of gradual restoration of male fertility in subsequent backcross generations was observed. Pollen from the fertile revertants did not transmit fertility restoration in progeny from crosses with the original CMS line and was poorly transmitted in sib-crosses. The appearance of fertile revertants and the different reactions of different CMS lines with the same cytoplasm in test-crosses may be caused by the action of recessive nuclear genes of the recurrent male parents that were accumulated during backcrossing; these may induce changes in cytoplasmic genes controlling CMS. Received: 5 March 1998 / Accepted: 7 April 1998     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

A CAPS marker associated with the partial restoration of cytoplasmic male sterility in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS), an economically important trait for hybrid seed production in many crops, is a maternally inherited trait in which a plant fails to produce functional anthers, pollen grains, or male gametes. It has long been reported that the restoration of CMS in chili pepper is controlled by a major nuclear gene termed restorer-of-fertility (Rf), along with several modifiers and some environmental factors. In this study, we identified the partial restoration (pr) locus related to the fertility restoration of CMS, demonstrated the inheritance of the trait, and developed a CAPS marker closely linked to the locus. The partially restored plant had normal anthers that produced a mix of normal and aborted pollen grains that stuck tightly to the anther wall, even after dehiscence. This trait was expressed only when the pepper plant had the sterile (S) cytoplasm and homozygous recessive pr alleles. A total of 768 AFLP primer combinations were screened, and bulked segregant analysis (BSA) was performed by preparing two pools of eight Pr/Pr (fully fertile) and eight pr/pr (partially fertile) plants, respectively, selected from the 87 individuals of the F₂ segregating population. Of the eight Pr-linked AFLP markers that were identified, E-AGC/M-GCA₁₂₂ and E-TCT/M-CCG₁₁₆ were the closest to the locus, estimated at about 1.8 cM in genetic distance. E-AGC/M-GCA₁₂₂ was converted into a CAPS marker, PR-CAPS, based on the sequences of the internal and flanking regions of the AFLP fragment. This PR-CAPS marker could be useful in selecting fully fertile lines (Pr/Pr) and eliminating partially fertile (pr/pr) and potential (Pr/pr) lines in segregant populations during the development of new inbred restorer lines.     

Mitochondrial and total DNA RAPD patterns can distinguish restorers of CMS lines in sorghum

 Seven sorghum restorer lines that differentially restore (or maintain) the A1 and A2 cytoplasmic male-sterile (CMS) cytoplasms were studied by RFLP analyses of their mtDNAs and RAPD analyses of their mitochondrial DNA (mtDNA) and total DNA to understand nuclear mitochondrial combinations that are present in these lines. RFLP data from 11 mitochondrial gene probes were inadequate to classify these seven lines. However, the analysis of RAPD profiles of total DNA could distinguish these lines on the basis of their ability to restore completely or partially the fertility in the A1/A2 CMS cytoplasms. Interestingly, RAPD profiles of mtDNAs of these lines also followed the same pattern as that of the total DNA. These results indicate that the different restorer lines possess specific nuclear-cytoplasm combinations. Further, the results also show that the RAPD technique can be used to identify markers for different cytoplasms used in CMS. Received: 26 August 1997 / Accepted: 9 October 1997     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

水稻细胞质雄性不育系及其保持系幼穗发育过程中内源激素的变化

利用酶联免疫测定技术研究了水稻细胞质雄性不育系珍汕97A及其保持系珍汕97B在幼穗发育过程中叶片、幼穗和花药中内源IAA、GA1＋4、ABA和iPAs含量的动态变化。结果表明,1）从幼穗发育的雌雄蕊形成期到三核花粉期,保持系叶片中IAA水平高于不育系,并在二核花粉期最高;在幼穗和花药中也以保持系为高。2）保持系与不育系叶片中GA1＋4含量变化趋势相似,均为先升后降,但从单核到三核花粉期以保持系为高;在幼穗和花药中也都以保持系为高。3）不育系叶片中ABA水平在幼穗发育早期明显高于保持系,中期与保持系相近,后期又高于保持系;在幼穗和花药中也都以不育系为高。4）保持系叶片、幼穗和花药中iPAs含量始终显著高于不育系。5）保持系叶片中IAA＋GA1＋4＋iPAs与ABA之比值也始终高于不育系。提示不育系叶片、幼穗和花药中IAA、GA1＋4、iPAs和ABA含量出现了异常,且IAA、GA1＋4和iPAs亏缺以及ABA盈积可能与水稻细胞质雄性不育发生有关。