Fine scale spatial structuring of sex and mitochondria in Silene vulgaris

Fine scale spatial structure (FSSS) of cytoplasmic genes in plants is thought to be generated via founder events and can be amplified when seeds germinate close to their mother. In gynodioecious species these processes are expected to generate FSSS in sex ratio because maternally inherited cytoplasmic male sterility genes partially influence sex expression. Here we document a striking example of FSSS in both mitochondrial genetic markers and sex in roadside populations of Silene vulgaris. We show that in one population FSSS of sexes influences relative fruit production of females compared to hermaphrodites. Furthermore, FSSS in sex ratio is expected to persist into future generations because offspring sex ratios from females are female-biased whereas offspring sex ratios from hermaphrodites are hermaphrodite-biased. Earlier studies indicated that pollen limitation is the most likely mechanism underlying negative frequency dependent fitness of females. Our results support the theoretical predictions that FSSS in sex ratio can reduce female fitness by decreasing the frequency at which females experience hermaphrodites. We argue that the influence of FSSS on female fitness is complementary to the influence of larger scale population structure on female fitness, and that population structure at both scales will act to decrease female frequencies in gynodioecious species. Better comprehension of the spatial structure of genders and genes controlling sex expression at a local scale is required for future progress toward understanding sex ratio evolution in gynodioecious plants.     

A Cytoplasmic Male Sterility-Associated Rearrangement of the Mitochondrial cobGene Region in Sugar Beet Beta vulgarisL.

Physical maps of the cobmtDNA region were constructed and compared between sugar beet Beta vulgarisL. plants with normal fertility and with cytoplasmic male sterility (CMS). A CMS-associated rearrangement did not affect the coding region of coband combined two mtDNA regions which are normally about 150 kb apart. Two point substitutions were found in the 3"-untranslated region of cob.     

水稻线粒体基因的表达受核背景的影响

高等植物细胞核与线粒体之间的互作机理一直是发育生物学的研究热点,而普遍存在于高等植物中的细胞质雄性不育现象的发现,为该领域的研究提供了极好的材料。以红莲型细胞质雄性不育系A、保持系B和两种杂交一代（F1和SF1）为材料,利用一种适用于线粒体基因表达分析的差异展示方法,研究了不同核背景下线粒体的变化情况。结果表明,核背景的改变对线粒体基因的表达具有广泛的影响。     

红麻线粒体中细胞质雄性不育候选基因cox3的克隆与分析

目的:比较红麻不育系和保持系线粒体基因组的差异,并克隆红麻细胞质雄性不育候选基因cox3,揭示红麻细胞质雄性不育的分子机理。方法:用Southern印迹方法研究红麻保持系和不育系线粒体基因组的差异;用同源克隆的方法克隆cox3基因。结果:不育系和保持系基因组存在较大差异;在保持系和不育系中克隆了cox3基因,其基因CDS区完全一致,基因长度为798 bp,GenBank序列号为HM535784;cox3基因与其他物种的cox3基因的同源性大于95.9%。结论:cox3基因的组织形式在不育系和保持系中存在差异,研究结果为揭示红麻细胞质雄性不育的机理提供了一定的依据。     

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids

We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped. Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.     

urf13-T of T cytoplasm maize mitochondria encodes a 13 kD polypeptide

Polyspecific antibody to a 17 amino acid synthetic peptide from the maize T-cytoplasm urf13-T mitochondrial open reading frame immunoprecipitated a 13 kD polypeptide from ³⁵S-methionine incorporations of T cytoplasm maize. Male-fertile, toxin-insensitive mutants in which the urf13-T sequence is deleted do not synthesize the 13 kD polypeptide. A mutant designated T-4, which carries a 5 bp insertion and a premature stop codon, synthesizes a truncated polypeptide, corresponding to an open reading frame of 8.3 kD. Thus the 13 kD polypeptide is trunctated or absent in mutants expressing male fertility and toxin insensitivity in T-cytoplasm maize.USDA-ARS     

Protein synthesis in mitochondria purified from roots, leaves and flowers of sugar beet

Highly purified, intact and functional mitochondria were isolated from roots and leaves of a number of fertile and male-sterile lines of sugar beet ( Beta vulgaris L.). Intact and functional mitochondria were successfully isolated from the flowers of fertile plants, but not from the flowers of male-sterile plants. Several alternative methods for the homogenization of male-sterile flowers were tried. Their failure suggests that the mitochondria from male-sterile flowers are more sensitive to mechanical damage than mitochondria from fertile, or other organs of male-sterile, plants.
In organello protein synthesis was optimized with respect to the total concentration of amino acids, the concentration of [³⁵S]-methionine, pH and respiratory substrate. Inhibitor experiments showed that the mitochondrial preparations contained mitochondrial translational activity only. With the exception of one band, no processing or proteolytic breakdown in either root or leaf mitochondrial protein synthesis products could be detected in pulse-chase experiments. Submitochondrial fractionation experiments showed the presence of two soluble polypeptides, whereas all other polypeptides were membrane bound.
The polypeptide patterns of root, leaf and flower mitochondria were very similar with the exception of 4 polypeptides involved in glycine oxidation. These 4 polypeptides were present in large amounts in leaf mitochondria and just detectable in flower mitochondria. The patterns of polypeptides syntesized in mitochondria isolated from roots, leaves and flowers also showed a number of organ-specific differences. Six qualitative and 6 quantitative differences were found between mitochondria isolated from these three organs. No unique polypeptides were found to be synthesized either by flower mitochondria or by mitochondria from roots and leaves of male-sterile plants compared to their male-fertile counterparts.     

甘蓝型油菜pol CMS育性恢复基因对orf224/atp6的转录调控

用 10个线粒体基因探针对波里马细胞质雄性不育 (polimaCMS)三系 1141A(pol) ,1141B(nap)和 1141R(pol)的花蕾线粒体RNA进行了Northern检测。结果表明 ,只有 3个探针atp6、orf2 2 4和orf2 2 2检测到转录本的差异。atp6在可育的 1141B中只转录产生一个丰度很高的 1 1kb转录本 ,在雄性不育的 1141A和pol胞质恢复系 1141R中 ,这个转录本的丰度明显减少并出现了分子量较大的 2个转录本 2 2kb、1 9kb转录本。与 1141A相比 ,恢复系1141R的 2 2kb和 1 9kb转录本丰度明显减少 ,并伴随着两个新的转录本 1 4kb和 1 3kb。表明orf2 2 4 atp6的表达与polCMS有关 ,并且其转录受到恢复基因Rfp的调控。同时通过对杂种F1 ( 1141A× 1141R)与另一个恢复系RS35 (pol)的比较证实 ,Rfp对orf2 2 4 atp6的调控与Rfp纯合与否无关。orf2 2 4 atp6在 1141A的苗期叶片中还转录产生育性恢复特异的 1 4kb转录本 ,这可能与细胞核基因型和相对低温条件有关。     

Gamma carbonic anhydrases in plant mitochondria

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s11103-005-5514-7     

植物细胞质雄性不育及其育性恢复的分子基础

植物细胞质雄性不育是广泛存在于高等植物中的现象,其表现为母性遗传、花粉败育,但雌蕊正常。细胞质雄性不育在杂交种子生产中起着重要作用,研究其分子作用机制有利于更有效地利用细胞质雄性不育。随着一些不育基因和恢复基因相继被克隆,人们对一些细胞质雄性不育和恢复系统的分子作用机理已经有一定了解。本文综述了近年来对植物细胞质雄性不育基因和恢复基因作用机理研究的进展。     

10个线粒体基因在甘蓝型油菜NCa不育系统不同组织中的转录调控及其与育性的关系

用10个线粒体基因为探针,对NCα不育系、保持系和可育F1的苗期叶片、幼蕾及未成熟种子的线粒体RNA进行了Northern分析。结果表明,这10个线粒体基因除atp6外,其余9个基因在同一材料的不同组织中没有表达差异,都属于组成型表达的线粒体基因。其中,off139、orf222、atp1、cox1、cox2、cob、rm5S、rm26S等8个线粒体基因在不育系、保持系和可育F1的苗期叶片、幼蕾及未成熟种子中有着相同的表达,属于表达不受核基因型影响,没有组织特异性的类型：atp9基因分别在同一材料的不同组织中的转录也基本没有变化,但是在3个不同的材料间具有表达差异：可能属于表达受核基因型影响、没有组织特异性的线粒体基因。atp6基因也在3个材料的叶、蕾和种子中都产生相同大小的转录本,但是在各个材料的不同组织中存在着信号强度的差异,可能是属于表达既受核基因型影响、又有组织特异性的线粒体基因。Orf222和off139分别在不育系和可育F1幼蕾中产生相同大小和丰度的转录本,但是在保持系幼蕾中没有检测到转录本;orf222检测到的3条转录本分别为1．1kb、0．9kb、0．6kb,而off139检测到0．8kb和0．6kb两条带。atp9探针在不育系和保持系幼蕾中都检测到1条0．6kb的转录本,而在可育F1幼蕾中检测到0．6kb和1．2kb的转录本。讨论了orf222、off139、atp9基因的表达与NCα细胞质雄性不育的可能关系。     

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

植物细胞质雄性不育及其育性恢复的分子生物学研究进展

植物细胞质雄性不育（CMS）和恢复系统在作物杂交种子生产中具有重要的意义。综述了目前已发现的与植物CMS相关的线粒体DNA位点,育性恢复基因对CMS相关DNA位点表达的影响,育性恢复基因的分子标记定位、克隆,及育性恢复分子机理等方面的研究进展,并讨论了恢复基因在植物分子育种上的应用。     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.