Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.     

一种新型无花瓣油菜突变不育系花器官的形态解剖研究

在自育的甘蓝型油菜(Brassica napus)无花瓣品系AP197中, 发现并育成由4对隐性核基因控制的无花瓣油菜突变不育系AMS971。AMS是一种雄蕊心皮化为不结实的假性雌蕊而引起的新的雄性不育类型, 不育性非常彻底, 其植株形态特征与AP197相同。比较花器官发现, AMS971除一个正常的雌蕊外,还有6个梭形的心皮化假性雌蕊。假心皮化雌蕊上部变成近似倒U形的柱头结构区, 下部是完全突变的半开裂心皮, 其上着生4~14个不等的裸露的幼小胚珠; 蜜腺比正常油菜小, 数目为0~4个, 这与AP197没有差异。AMS971的四轮花器官由于突变造成了大小和重量的重新分配。推断AMS为类似拟南芥属B功能缺失的突变体。     

<Emphasis Type="Italic">Brassica napus</Emphasis> lines with rearranged <Emphasis Type="Italic">Arabidopsis</Emphasis> mitochondria display CMS and a range of developmental aberrations

Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC(3) and BC(6) generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC(5) generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.     

无花瓣油菜雄蕊心皮化突变体细胞学观察及基因表达分析

油菜是我国重要的油料作物,油菜花器官具有典型的十字花科特点,无花瓣油菜在花期不存在花冠层,这种特点有助于提高油菜产量,预防茵核病的传播。雄蕊心皮化是指花器官的雄蕊结构被具有类似于雌蕊结构的器官代替,这不仅造成了花器官结构的变化也导致了雄性不育。本文通过对无花瓣油菜雄蕊心皮化突变不育分离群体中的雄性可育株和不育株的比较研究,发现心皮化现象是由遗传因素引起的。细胞学观察发现,雄蕊心皮化在花器官发育的早期就已经产生,心皮化的雄蕊中着生类似于胚珠的结构,其顶端细胞的形态和排列方式也与雌蕊柱头相似。花发育相关基因的表达分析表明,B组基因彳丹在不育株3轮花器中的表达都比可育株低,特别是在第二轮花器官中这种差异最为突出。而A组基因AP1在不育株第二轮花器官中的表达量较可育株高。c组基因AGL8、SHPI、SHP2、NAP在不育株心皮化的第二轮花器官中表达都较可育株中高。     

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

The FLO10 Gene Product Regulates the Expression Domain of Homeotic Genes AP3 and PI in Arabidopsis Flowers

We describe a novel mutant of Arabidopsis, Flo10, which is the result of a recessive allele, flo10, in the nuclear gene FLO10. The first three organ whorls (sepals, petals, and stamens) of Flo10 flowers are normal, but the fourth, gynoecial whorl is replaced by two to eight stamens or stamen-carpel intermediate organs. Studies of ontogeny suggest that the position of the first six of these fourth-whorl organs often resembles that of the wild-type third-whorl organs. To determine the interaction of the FLO10 gene with the floral organ homeotic genes APETALA2 (AP2), PISTILLATA (PI), AP3, and AGAMOUS (AG), we generated lines homozygous for flo10 and heterozygous or homozygous for a recessive allele of the homeotic genes. On the basis of our data, we suggest that FLO10 functions to prevent the expression of the AP3/PI developmental pathway in the gynoecial (fourth) whorl.     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

细胞质雄性不育水稻线粒体基因组的RFLP分析

利用RFLP技术,比较研究了在农业生产上广泛应用的9种细胞质雄性不育体系的线粒体基因组,结果表明：1）9种水稻雄性不育细胞质的遗传相似性变化范围为0．615～1．000。所有配子体雄性不育细胞质的遗传相似性变化范围为0．6431．000,其中QXA,DY1A和YM15A这3种细胞质的遗传相似性为1．000。所有孢子体雄性不育细胞质的遗传相似性为1．000,2）在配子体细胞质雄性不育水稻中,用3个探针（atpa,atp9＋nad6,cox2）与2种内切酶（HindⅢ,BamHⅠ）的组合未发现不育系与保持系之间的多态性,但在探针atp6,cob,和had2的杂交带型中找到了不育系和保持系之间的一些差异。YTA和YTB在5个探针．内切酶组合（atp61HindⅢ,coblHindⅢ,atp61BamHⅠ,coblBamHⅠ,nad21BamHⅠ）中存在差异。3种细胞质（QX,SJ,DY1）的不育系和保持系之间的差异是相同的,都出现在atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ等3个组合中。YM15A和YM15B在4个组合（atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ,nad2／BamHⅠ）中存在差异。LYA和LYB的差异出现在cob／HindⅢ,cob／BamHⅠ,nad2／BamHⅠ这3个组合中;3）在孢子体细胞质雄性不育水稻中,所有不育系的带型是一样的,所有保持系的带型也一样。不育系和保持系的差异出现在atp6／HindⅢ,cob／HindⅢ,atp6／EcoR,Ⅰcob／EcoRⅠ,cox1／EcoRⅠ,atp6／BamHⅠ,cob／BamHⅠ,cox1／BamHⅠ,cox2／BamHⅠ等9个组合中。这些结果在分子水平上揭示了9种雄性不育细胞质的线粒体基因组存在结构多样性,并为其细胞质雄性不育分子机理的研究打下了基础。     

Isolation and characterization of a male sterility gene homolog <Emphasis Type="Italic">BcMS2</Emphasis> from Chinese cabbagge-pak-choi that expressing in an anther-specific manner

A male sterility gene homolog, designated BcMS2, was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533. Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from Brassica napus revealed some differences in gene structure and evolution. The full genomic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns, more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stage III flower buds of male fertile Chinese cabbage-pak-choi 'ZUBajh97-01B' and there were no detection in all organs of Polima cytoplasmic male sterility (CMS) line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'. Furthermore, RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals, petals, filaments and pistils. These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.