Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Effects of water stress on male gametophyte development in plants

 Male reproductive development in plants is highly sensitive to water deficit during meiosis in the microspore mother cells. Water deficit during this stage inhibits further development of microspores or pollen grains, causing male sterility. Female fertility, in contrast, is quite immune to stress. The injury is apparently not caused by desiccation of the reproductive tissue, but is an indirect consequence of water deficit in the vegetative organs, such as leaves. The mechanism underlying this stress response probably involves a long-distance signaling molecule, originating in the organs that undergo water loss, and affecting fertility in the reproductive tissue, which conserves its water status. Much research has been focused on the involvement of abscisic acid in this regard, but the most recent evidence tends to reject a role for this hormone in the induction of male sterility. Stress-induced arrest of male gametophyte development is preceded by disturbances in carbohydrate metabolism and distribution within anthers, and an inhibition of the key sugar-cleaving enzyme, acid invertase. Since invertase gene expression can be modulated by sugar concentration, it is possible that decreased sugar delivery to reproductive tissue upon inhibition of photosynthesis by stress is the signal that triggers metabolic lesions leading to failure of male gametophyte development. Received: 31 October 1996 / Revision accepted: 18 February 1997     

Cold-induced repression of the rice anther-specific cell wall invertase gene OSINV4 is correlated with sucrose accumulation and pollen sterility

Low temperatures during rice (Oryza sativa L.) pollen development cause pollen sterility and decreased grain yield. We show that the time of highest sensitivity to cold coincides with the time of peak tapetal activity: the transition of the tetrad to early uni-nucleate stage (young microspore, YM stage). Low temperatures at this stage of pollen development result in an accumulation of sucrose in the anthers, accompanied by decreased activity of cell wall bound acid invertase and depletion of starch in mature pollen grains. Expression analysis of two cell wall (OSINV1, 4) and one vacuolar (OSINV2) acid invertase genes showed that OSINV4 is anther-specific and down-regulated by cold treatment. OSINV4 is transiently expressed in the tapetum cell layer at the YM stage, and later from the early binucleate stage in the maturing microspores. The down-regulation of OSINV4 expression in the tapetum at YM may cause a disruption in hexose production and starch formation in the pollen grains. In a cold-tolerant cultivar, OSINV4 expression was not reduced by cold; sucrose did not accumulate in the anthers and starch formation in the pollen grains was not affected.     

Phenotype Analysis and Fine Mapping of the Male Sterile Mutant <i>ms10</i> in Rice (<i>Oryza sativa</i> L.)

There is a positive correlation between fertility and yield, and the decrease of fertility is bound to a greatly reduced crop yield. Male sterile mutants can be used in hybrid rice. Therefore, rice male sterility has an important value in research and application, and the study of related mutants is also very vital. The mutant ms10 (male sterile 10) reported in this study was induced by ethyl methane sulfonate (EMS) in the indica maintainer line Xinong 1B. There was no significant difference between the ms10 and wild type in the vegetative growth stage. However, in the reproductive growth stage, ms10 showed that the plant became shorter, the anther became smaller and the color became lighter, and finally showed the phenotype of male sterility in comparison to the wild type. I₂-KI staining showed that the pollen was malformed and only a little was active. Scanning electron microscopy observation showed that the exine waxy layer of the ms10 anther decreased, suggesting that the protective effect on pollen was decreased. This may be one of the reasons leading to the phenotype of male sterility. Finally, the pollen showed shrinkage and collapsed, and the structure of germinating pore cover disappeared. This may be the result of sterility. Genetic analysis showed that the male sterility phenotype of the mutant was controlled by a single recessive nuclear gene. MS10 was mapped between the molecular markers IND37 and IND51 on chromosome 4, with a physical distance of 178.6 kb. These results lay the foundation for further studies on MS10.     

Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.     

Extracellular invertase: key metabolic enzyme and PR protein

Extracellular invertase is the key enzyme of an apoplasmic phloem unloading pathway and catalyses the hydrolytic cleavage of the transport sugar sucrose released into the apoplast. This mechanism contributes to long-distance assimilate transport, provides the substrate to sustain heterotrophic growth and generates metabolic signals known to effect various processes of primary metabolism and defence responses. The essential function of extracellular invertase for supplying carbohydrates to sink organs was demonstrated by the finding that antisense repression of an anther-specific isoenzyme provides an efficient method for metabolic engineering of male sterility. The regulation of extracellular invertase by all classes of phytohormones indicates an essential link between the molecular mechanism of phytohormone action and primary metabolism. The up-regulation of extracellular invertase appears to be a common response to various biotic and abiotic stress-related stimuli such as pathogen infection and salt stress, in addition to specific stress-related reactions. Based on the observed co-ordinated regulation of source/sink relations and defence responses by sugars and stress-related stimuli, the identified activation of distinct subsets of MAP kinases provides a mechanism for signal integration and distribution within such complex networks. Sucrose derivatives not synthesized by higher plants, such as turanose, were shown to elicit responses distinctly different from metabolizable sugars and are rather perceived as stress-related stimuli.     

Ecological and genetic factors contributing to the low frequency of male sterility in Chamaecrista Fasciculata (Fabaceae)

Bumble bee pollinated Chamaecrista fasciculata provides pollen as the sole reward to its pollinators. Male sterility, expressed as an absence or nearly complete absence of pollen production, occurs in low frequency in populations of C. fasciculata. Here we describe experiments, using C. fasciculata, to examine frequently cited determinants of the spread and maintenance of male sterility: compensation and the genetic basis of male sterility. In addition, we examine the role the pollination system plays in determining the reproductive success of the male steriles. Seventeen populations in Maryland, Illinois, and Kansas were surveyed and found to range from 0 to 6% male sterility per population. An artificial population of male-sterile simulants and hermaphrodites was created to examine how the local frequency of nonrewarding male steriles might affect male-sterile female reproductive success. Male steriles performed equally poorly, with respect to seed production, whether surrounded by other male-sterile simulants or hermaphrodites. Compensation was examined by comparison of male steriles and hermaphrodites with respect to several reproductive and nonreproductive characters. Male steriles outperformed hermaphrodites in terms of nonreproductive biomass, but performed equally in terms of ovule number and produced many fewer flowers. The genetic basis of male sterility was examined by performing both intra- and interpopulational crosses of male steriles to hermaphrodites and indicate that male sterility is not purely cytoplasmic. The low frequency of male sterility in C. fasciculata populations may reflect reduced female reproductive success because of pollinator avoidance, lack of reproductive compensation, and a mode of inheritance that is not purely cytoplasmic.     

Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen

Male reproductive development of rice (Oryza sativa L.) is very sensitive to drought. A brief, transitory episode of water stress during meiosis in pollen mother cells of rice grown under controlled environmental conditions induced pollen sterility. Anthers containing sterile pollen were smaller, thinner, and often deformed compared to normal anthers of well-watered plants. Only about 20% of the fully developed florets in stressed plants produced grains, compared to 90% in well-watered controls. Water stress treatments after meiosis were progressively less damaging. Levels of starch and sugars and activities of key enzymes involved in sucrose cleavage and starch synthesis were analyzed in anthers collected at various developmental stages from plants briefly stressed during meiosis and then re-watered. Normal starch accumulation during pollen development was strongly inhibited in stress-affected anthers. During the period of stress, both reducing and non-reducing sugars accumulated in anthers. After the relief of stress, reducing sugar levels fell somewhat below those in controls, but levels of non-reducing sugars remained higher than in controls. Activities of acid invertase and soluble starch synthase in stressed anthers were lower than in controls at comparable stages throughout development, during as well as after stress. Stress had no immediate effect on ADP-glucose pyrophosphorylase activity, but had an inhibitory aftereffect throughout post-stress development. Sucrose synthase activity, which was, relatively speaking, much lower than acid invertase activity, was only slightly suppressed by stress. The results show that it is unlikely that pollen sterility, or the attendant inhibition of starch accumulation, in water-stressed rice plants are caused by carbohydrate starvation per se. Instead, an impairment of enzymes of sugar metabolism and starch synthesis may be among the potential causes of this failure.     

Antisense inhibition of a nuclear gene,BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment

Male sterility is widely used for the production of hybrid seeds, but the use of genic male sterility is rather limited because of difficulty in maintaining homozygous male sterile plants. Recently, the DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) gene, which encodes a phospholipase A1 involved in the first step of the jasmonic acid (JA) biosynthesis pathway, was isolated from a male sterile Arabidopsis mutant. To utilize this gene in Brassica crops, we characterized the BrDAD1 gene, the putative ortholog of DAD1 in Brassica rapa. Out of 25 plants transformed with an antisense gene constructed from the BrDAD1, 3 plants showed a defect of anther dehiscence at the flower bud opening stage and produced inviable pollen. One of the three showed male sterility only, but the other two showed a delay or a lack of flower opening in addition to male sterility. The male sterile and flower-opening phenotypes were rescued by the application of JA as well as linolenic acid. Furthermore, all these characteristics were inherited to the next generation. The present results demonstrate a novel control system for hybrid seed production by the use of nuclear genes.     

The effect of genotype on pollen transmission of mitochondria in rapeseed (Brassica napus)

Summary Crossing experiments were conducted to determine whether parental genotype affected the rate of transmission of paternal mitochondria to progeny in rapeseed (Brassica napus). Progeny were screened either by RFLP analysis of mitochondrial (mt) DNA or by means of a mt marker that causes male sterility. To date we have transferred paternal mitochondria to progeny in only cross, i.e. a specific female line crossed to a specific male line. The male line carries the polima cytoplasm, the mitochondria of which confer a characteristic malesterile flower morphology when in a napus nuclear background. This line is male fertile due to a restorer gene carried on an extra chromosome from a closely related species, Brassica juncea. The female line has a Brassica campestris cytoplasm with a chloroplast mutation conferring resistance to triazine herbicides. Progeny with mixtures of parental mtDNA display a range of plant phenotype from complete male fertility through varying proportions of male-sterile sectors to complete male sterility. The male sterility or fertility of flowers on a sector of a plant reflects the mt population of that sector, and such sectors will give rise to stably fertile or sterile progeny. These experiments suggest that maternal inheritance of mitochondria in higher plants is due to genes active in both the pollen parent and the egg parent.     

Analysis of gene expression profile in pollen development of recessive genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. line S45A

Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sicwinv1, a Cell Wall Invertase from Sesame,Is Involved in Anther Development

Journal of Plant Growth Regulation - Male sterility is an effective phenotype for sesame hybrid production. Cell wall invertase (CWINV) plays a crucial role in providing carbohydrates for male...     

Male sterility in triploid dandelions: asexual females vs. asexual hermaphrodites

Male reproductive output, pollen in plants and sperm in animals has been shown to constitute a substantial cost for many organisms. In parthenogenetic hermaphrodites, selection is therefore expected to reduce the allocation of resources to male reproductive output. However, sustained production of pollen or sperm has been observed in numerous asexual hermaphrodites. We studied the widespread production of pollen by triploid asexual dandelions, Taraxacum sect. Ruderalia, comparing rare male sterile individuals with pollen producing asexuals. We found that individuals can show plasticity in the production of pollen, but that it is nevertheless possible to distinguish between (facultatively) male sterile asexuals and male fertile asexuals. Based on evidence from genetic markers and crosses, we conclude that the male sterility in asexual dandelions is caused by nuclear genes, in contrast to the cytoplasmically inherited male sterility previously found in sexual dandelions. Male sterile lineages did not produce more seeds per flower head, heavier seeds or seeds that were more viable. However, male sterile plants did produce more seed heads and hence more seeds than pollen producing ones, indicating that they were able to reallocate resources toward seed production. Considering the difference in seed production, it remains puzzling that not more asexual dandelions are male sterile.     

Cytological investigation of male sterility in sorghum caused by a dominant mutation (<Emphasis Type="Italic">Ms</Emphasis><Subscript><Emphasis Type="Italic">tc</Emphasis></Subscript>) derived from tissue culture

Male sterility mutations are an important tool in the investigation of anther and pollen development and for obtaining hybrid seeds in plant breeding. Cytological analysis of microsporo- and microgameto-genesis in sorghum plants with dominant mutation of male sterility (Ms_tc) derived from tissue culture has been carried out. Using substitution backcrosses, this mutation was introduced first into the nuclear background of the fertile sorghum line SK-723 and from this line into Volzhskoe-4w (V-4w). The mechanism of Ms_tc action on anther and pollen development differed in different nuclear backgrounds. In SK-723, phenotypic expression of Ms_tc began before the beginning of meiosis, which resulted in degeneration of sporogenous tissue in some anthers and in significant disturbances of anther morphology. In microsporocytes that did not degenerate, the frequency of non-specific meiotic abnormalities characteristic of the fertile line SK-723 significantly increased. In addition, in the mutant plants, a number of specific meiotic abnormalities--almost complete desynapsis, and formation of syncytial structures--were observed, apparently the consequence of Ms_tc action. In mono- or bi-nucleate microspores, degenerative processes resulting in formation of empty or anomalously coloured pollen grains led to almost complete male sterility. In the V-4w nuclear background, changes in anther structure and meiotic disturbances were infrequent. The degenerative processes began at the uni- or binucleate microspore stage and resulted in formation of empty or abnormally coloured pollen grains, and in partial pollen sterility. Thus, the same nuclear male sterility-inducing mutation in different nuclear backgrounds affects different stages of pollen development.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.