Efficient production of genetically engineered,male-sterile <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> using anther-specific promoters and genes derived from <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">B. rapa</Emphasis>

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

羽叶薰衣草雄性不育的细胞学研究

用光镜和电镜观察羽叶薰衣草(Lavandula pinnata L.)雄性不育小孢子发育过程的细胞形态学特征.结果表明:羽叶薰衣草花药4枚,每枚花药通常具4个小孢子囊.花药壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四面体及十字交叉型.小孢子的发育过程可分为造孢细胞期、减数分裂时期、小孢子发育早期、小孢子发育晚期.未观察到二胞花粉期和成熟花粉期.羽叶薰衣草花粉败育主要发生在单核花粉时期,细胞内物质解体并逐渐消失变成空壳花粉或花粉皱缩变形成为各种畸形的败育花粉.在此之前小孢子的发育正常.羽叶薰衣草小孢子不育机制体现在绒毡层过早解体、四分体时期以后各细胞中线粒体结构不正常、胼胝质壁与小孢子母细胞脱离、花药壁细胞中淀粉出现时间异常等. 壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四面体及十字交叉型.小孢子的发育过程可分为造孢细胞期、减数分裂时期、小孢子发育早期、小孢子发育晚期.未观察到二胞花粉期和成熟花粉期.羽叶薰衣草花粉败育主要发生在单核花粉时期,细胞内物质解体并逐渐消失变成空壳花粉或花粉皱缩变形成为各种畸形的败育花粉.在此 前小孢子的发育正常.羽叶薰衣草小孢子不育机制体现在绒毡层过早解体、四分体时期以后各细胞中线粒体结构不正常、胼胝质壁与小孢子母细胞脱离、花药壁细胞中淀粉出现时间异常等. 壁发育为双子叶型,从外向内分为表皮、药室内壁、中层和绒毡层4层细胞.减数分裂形成的四分体为四     

长春蒲公英雄性不育株形态学观察与败育细胞学研究

在长春蒲公英(Taraxacum junpeianum Kitam.)株群中发现雄性不育现象,为研究其败育机理及特点,探寻其不育基因,采用形态观察法、石蜡切片技术和染色体压片法,对长春蒲公英野生型及其雄性不育株的花药发育过程和花粉母细胞减数分裂过程进行了观察。结果表明:(1)长春蒲公英雄性不育株花药中部发红、干瘪、无花粉散出。与野生型比较,雄性不育株雄蕊更短,子房更窄,种子形态更加狭长;(2)长春蒲公英雄性不育株败育时期为四分体到单核小孢子前期,败育方式为小孢子自身异常发育,绒毡层异常分解,互相粘连败育;(3)长春蒲公英雄性不育株花粉母细胞减数分裂二分体时期出现落后微核,随后产生极少四分体,并且四分体产生大量染色体桥,小孢子营养物质流失,彻底败育。因此,长春蒲公英雄性不育株败育彻底、稳定,并且有种的特点。小孢子自身异常发育和绒毡层异常分解是导致败育的主要原因。     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

云南松小孢子发生过程中绒毡层细胞的超微结构研究

云南松(Pinus yunnanensis Fr.)在小孢子囊发育早期,绒毡层原生质体发生收缩,并伴随着细胞壁厚度的增加。脂肪微滴和孢粉素物质沉积而形成周缘绒毡层膜。随着孢粉素物质的产生,绒毡层细胞质明显地液泡化。孢粉素物质在绒毡层细胞膨大的内质网槽库中形成,随后被排放到近邻的小液泡内或游离于细胞质中。孢粉素物质也可在特化的含片层的质体中形成,孢粉素依附在片层膜上,或释放到细胞质中。     

Strategies for retinal design in arthropod eyes of low F-number

Summary The superposition eyes and simple eyes of many arthropods have apertures (A) with a diameter bigger than, or about the same size as, the focal length of the eye (f). That is, these eyes have low F-numbers (f/A). Many of the light rays focussed onto a photoreceptor will not be trapped by total internal reflection in the photoreceptor and will therefore pass through and be absorbed in photoreceptors other than that for which the light was intended. This spread of light in the retina leads to a broadening of the angular-sensitivity function and a consequent degrading of the image at the retinal level. A number of solutions to this problem are found in nature, with the most effective that of isolating the photoreceptors with a sheath of either light-absorbing pigment or reflecting tapetum. A ray-tracing model was used to assess the relative merits of the tapetal and pigment sheath designs in low F-number superposition eyes, and also to investigate the effect of changing the refractive index and absorption coefficient of the rhabdom. Which sheathing solution is best depends on the quality of the image on the retina, on the spacing of the rhabdoms in the retina and on the intensity of light normally experienced by the eye. In a retina with closely packed rhabdoms, the model predicts full sheathing to be the optimal solution if the image is well-focussed, partial sheathing if poorly focussed and no sheathing if moderately well-focussed. In a retina with rhabdoms spaced apart and a well-focussed image, the model predicts partial sheathing to be optimal. A pigment sheath is predicted to be useful in eyes which experience bright light and have no need for high sensitivity. A tapetal sheath is predicted to be useful at any intensity. A survey of arthropod eyes with low F-number supports the predictions of the model.     

Anther-specific expression of mutated melon ethylene receptor gene Cm-ERS1/H70A affected tapetum degeneration and pollen grain production in transgenic tobacco plants

To develop a new system for inducible male sterility without any modification of the floral architecture in tobacco plants, a mutated ethylene receptor gene Cm-ERS1/H70A was fused either to the tobacco Nin88 promoter known to function mainly in the tapetum and microspore or to the CaMV 35S promoter known to be a constitutive promoter. The fusion genes pNin88::Cm-ERS1/H70A and p35S::Cm-ERS1/H70A were introduced in tobacco plants, which generated two independent transformants. Transformants with 35S::Cm-ERS1/H70A produced less normal pollen and had modified floral architecture while those with Nin88::Cm-ERS1/H70A produced less normal pollen without modification of floral architecture. Histological observations of anthers at stage 2 showed that tapetum degeneration in NH70A #8 and H70A #2 transformants occurred later than in wild types, strongly indicating that the expression of the mutated gene was involved in this delay. These results suggest that the tapetum-specific expression of a mutated ethylene receptor gene is a potential strategy for inducing male sterility in transgenic plants.     

麦冬花药绒毡层和乌氏体的细微结构

麦冬(Ophiopogon japonicus)的绒毡层发育为分泌型。在小孢子母细胞时期,绒毡层细胞达到了发育的高峰。此时,绒毡层细胞中细胞器非常丰富,具大量线粒体、高尔基体和质体,尤以肉质网含量最多;原乌氏体出现较早,在小孢子母细胞时期绒毡层细胞中就已出现;四分体时期,大量原乌氏体被排入内切向面的质膜和纤维素壁之间;到了小孢子早期,绒毡层细胞失去细胞壁,原乌氏体分布在质膜的凹陷处,孢粉素物质在其上沉积,发育为乌氏体,乌氏体有单个和复合两种类型;当花粉成熟时,绒毡层细胞完全解体。     

Effects of copper deficiency on pollen fertility and nucleic acids in the durum wheat anther

Summary The effects of continuous copper deficiency and of temporary deficiencies initiated prior to (initial deficiency) or after (terminal deficiency) the end of tillering (a stage preceding the meiotic cycle) on pollen formation were investigated in durum wheat grown on a nutrient solution. The effects of the treatments on pollen viability (FCR test) and on the proline content of the pollen grains suggested that copper deficiency induced a nearly complete sterility of the pollen formed and inhibited all grain production. Temporary copper deficiencies significantly reduced the viability rate and the number of proline-rich pollen grains without affecting pollengrain production. Cytophotometric measurements showed that initial copper deficiency induced a significant increase in the proportion of polyploid microspore mother cells (MMCs), whereas terminal copper deficiency blocked endomitotic DNA syntheses. Protein metabolism was markedly altered by the treatments. The RNA content of the cytoplasm of tapetum cells was decreased by 34%–48%, depending on the treatment. The autoradiographic study showed that the stress caused by copper deficiency enhanced [³H]uridine incorporation into microspore cytoplasm RNA and also into the tapetum cells in the case of temporary deficiencies. The incidence of the treatments on the ploidy of the mother cells and on the disturbance of protein metabolism, particularly in tapetum cells, is discussed.     

ABA regulates apoplastic sugar transport and is a potential signal for cold-induced pollen sterility in rice

Cold temperatures cause pollen sterility and large reductions in grain yield in temperate rice growing regions of the world. Induction of pollen sterility by cold involves a disruption of sugar transport in anthers, caused by the cold-induced repression of the apoplastic sugar transport pathway in the tapetum. Here we demonstrate that the phytohormone ABA is a potential signal for cold-induced pollen sterility (CIPS). Cold treatment of the cold-sensitive cultivar Doongara resulted in increased anther ABA levels. Exogenous ABA treatment at the young microspore stage induced pollen sterility and affected cell wall invertase and monosaccharide transporter gene expression in a way similar to cold treatment. In the cold-tolerant cultivar R31, ABA levels were significantly lower under normal circumstances and remained low after cold treatment. The differences in endogenous ABA levels in Doongara and R31 correlated with differences in expression of the ABA biosynthetic genes encoding zeaxanthin epoxidase (OSZEP1) and 9-cis-epoxycarotenoid dioxygenase (OSNCED2, OSNCED3) in anthers. The expression of three ABA-8-hydroxylase genes (ABA8OX1, 2 and 3) in R31 anthers was higher under control conditions and was regulated differently by cold compared with Doongara. Our results indicate that the cold tolerance phenotype of R31 is correlated with lower endogenous ABA levels and a different regulation of ABA metabolism.