Sterility In Transgenic Plants Research Articles

Overexpression of sesame polyketide synthase A leads to abnormal pollen development in Arabidopsis

BackgroundSesame is a great reservoir of bioactive constituents and unique antioxidant components. It is widely used for its nutritional and medicinal value. The expanding demand for sesame seeds is putting pressure on sesame breeders to develop high-yielding varieties. A hybrid breeding strategy based on male sterility is one of the most effective ways to increase the crop yield. To date, little is known about the genes and mechanism underlying sesame male fertility. Therefore, studies are being conducted to identify and functionally characterize key candidate genes involved in sesame pollen development. Polyketide synthases (PKSs) are critical enzymes involved in the biosynthesis of sporopollenin, the primary component of pollen exine. Their in planta functions are being investigated for applications in crop breeding.ResultsIn this study, we cloned the sesame POLYKETIDE SYNTHASE A (SiPKSA) and examined its function in male sterility. SiPKSA was specifically expressed in sesame flower buds, and its expression was significantly higher in sterile sesame anthers than in fertile anthers during the tetrad and microspore development stages. Furthermore, overexpression of SiPKSA in Arabidopsis caused male sterility in transgenic plants. Ultrastructural observation showed that the pollen grains of SiPKSA-overexpressing plants contained few cytoplasmic inclusions and exhibited an abnormal pollen wall structure, with a thicker exine layer compared to the wild type. In agreement with this, the expression of a set of sporopollenin biosynthesis-related genes and the contents of their fatty acids and phenolics were significantly altered in anthers of SiPKSA-overexpressing plants compared with wild type during anther development.ConclusionThese findings highlighted that overexpression of SiPKSA in Arabidopsis might cause male sterility through defective pollen wall formation. Moreover, they suggested that SiPKSA modulates vibrant pollen development via sporopollenin biosynthesis, and a defect in its regulation may induce male sterility. Therefore, genetic manipulation of SiPKSA might promote hybrid breeding in sesame and other crop species.

A Novel R2R3-MYB Gene LoMYB33 From Lily Is Specifically Expressed in Anthers and Plays a Role in Pollen Development.

Lily (Lilium spp.) is an important commercial flower crop, but its market popularity and applications are adversely affected by severe pollen pollution. Many studies have examined pollen development in model plants, but few studies have been conducted on flower crops such as lily. GAMYBs are a class of R2R3-MYB transcription factors and play important roles in plant development and biotic resistance; their functions vary in different pathways, and many of them are involved in anther development. However, their function and regulatory role in lily remain unclear. Here, the GAMYB homolog LoMYB33 was isolated and identified from lily. The open reading frame of LoMYB33 was 1620 bp and encoded a protein with 539 amino acids localized in the nucleus and cytoplasm. Protein sequence alignment showed that LoMYB33 contained a conserved R2R3 domain and three BOX motifs (BOX1, BOX2, and BOX3), which were unique to the GAMYB family. LoMYB33 had transcriptional activation activity, and its transactivation domain was located within 90 amino acids of the C-terminal. LoMYB33 was highly expressed during the late stages of anther development, especially in pollen. Analysis of the promoter activity of LoMYB33 in transgenic Arabidopsis revealed that the LoMYB33 promoter was highly activated in the pollen of stage 12 to 13 flowers. Overexpression of LoMYB33 in Arabidopsis significantly retarded growth; the excess accumulation of LoMYB33 also negatively affected normal anther development, which generated fewer pollen grains and resulted in partial male sterility in transgenic plants. Silencing of LoMYB33 in lily also greatly decreased the amount of pollen. Overall, our results suggested that LoMYB33 might play an important role in the anther development and pollen formation of lily.

Fine regulation of ARF17 for anther development and pollen formation

BackgroundIn Arabidopsis, the tapetum and microsporocytes are critical for pollen formation. Previous studies have shown that ARF17 is expressed in microsporocytes and tetrads and directly regulates tetrad wall synthesis for pollen formation. ARF17 is the direct target of miR160, and promoterARF17::5mARF17 (5mARF17/WT) transgenic plants, which have five silent mutations within the miR160-complementary domain, are sterile.ResultsHere, we found that ARF17 is also expressed in the tapetum, which was defective in arf17 mutants. Compared with arf17 mutants, 5mARF17/WT plants had abnormal tapetal cells and tetrads but were less vacuolated in the tapetum. Immunocytochemical assays showed that the ARF17 protein over-accumulated in tapetum, microsporocytes and tetrads of 5mARF17/WT plants at early anther stages, but its expression pattern was not affected during anther development. 5mARF17 driven by its native promoter did not rescue the arf17 male-sterile phenotype. The expression of 5mARF17 driven by the tapetum-specific promoter A9 led to a defective tapetum and male sterility in transgenic plants. These results suggest that the overexpression of ARF17 in the tapetum and microsporocytes of 5mARF17/WT plants leads to male sterility. Microarray data revealed that an abundance of genes involved in transcription and translation are ectopically expressed in 5mARF17/WT plants.ConclusionsOur work shows that ARF17 plays an essential role in anther development and pollen formation, and ARF17 expression under miR160 regulation is critical for its function during anther development.

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.

A novel herbicide‐inducible male sterility system

Heterosis is a phenomenon that first-generation offspring perform better than their parents. Conventional breeding methods have their shortcomings. It would be optimal to construct inducible male sterile plants. We developed a novel system for creating male sterile transgenic plants by downregulating the specific expression of the glyphosate tolerance CP4 EPSPS gene in male reproductive tissues. Transcriptional repression was achieved by manipulating DNA binding proteins with their specific corresponding sites. We transferred the CP4 EPSPS gene driven by a modified CaMV 35S promoter with three tetracycline operator copies in the vicinity of the TATA box and tetracycline repressor gene under the control of an anther-specific promoter Osg6B to Arabidopsis thaliana. As a result, we successfully obtained controllable transgenic plants: the whole plant could tolerate exposure of glyphosate but the male tissue was sensitive. The novel inducible male sterility system is applied and easy to handle, so it might be applicable to a wide range of crop plants.

Development of adjustable male sterile plant in broccoli by antisense DAD1 fragment transformation

Male sterile line is very important in hybrid seed production in broccoli (Brassicaoleracea L. var. italica). A fragment of BoiDAD1F was amplified from this specie using primers designed against the conserved DAD1F fragment of DAD1 genes inArabidopsis thaliana and Brassica rapa. The BoiDAD1F sequence was 88 and 99% similar to the homologous genes in Arabidopsis and B. rapa, respectively. Theantisense BoiDAD1F was then cloned onto pBI 121 vector to make the transformation construct, pBI-antiBoiDAD1F. Cotyledons with petiole were inoculated with Agrobacterium tumefaciens harboring the pBI-antiBoiDAD1F plasmid. Positive transformation was confirmed using polymerase chain reaction (PCR) and Southern blots. Twenty individual transformants were obtained, of which 15 showed suppressed DAD1 mRNA level. Pollens produced by the male sterile transgenic plants germinated poorly (≤ 10%). No seeds were produced on the flowering transgenic plants, and the siliquae were empty, or the few shrinked seeds, which failed to germinate. When pollinated with wild type plant pollen, the siliquas of the transgenic plants enlarged to produce normal seeds. No morphological differences were observed between the transgenic and wild type plants, except a few abnormal flowers on transgenic ones. To restore the fertility, exogenous JA were applied by dipping the floral buds into an aqueous solution containing 500 μM MeJA and 0.05% Tween 20 after removal of the opened flowers on the inflorescence. The treated flower produced normal pollens which germinated on the medium, and were fertile. T1 population consisted of fertile and sterile plants in the ratio of 3:1. The T2 population comprised of both complete fertile and sterile lines. These results confirmed the Mendel model of separation of the antisense RNA insert in the transgenic plants. Key words: Brassica oleracea L. var. italica, DAD1, adjustable male sterility, jasmonic acid and antisense RNA.

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence

Plant mitochondria include gamma-type carbonic anhydrases (gammaCAs) of unknown function. In Arabidopsis, the gammaCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2-3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H(2)O(2) dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.

Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco

To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3'end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (-60) minimal promoter [AG-I-35S (-60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (-60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (-60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (-60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering.

RNAi-mediated male sterility of tobacco by silencing TA29

The superior performance of F1 hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.

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.

Production of N-Acetyl-Phosphinothricin: A Substance used for Inducing Male Sterility in Transgenic Plants

The non-toxic compound N-acetyl-L-phosphinothricin (N-Ac-L-PPT) is used in a so-called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L-phosphinothricin (L-PPT). A procedure was developed to produce pure racemic and L-isomeric N-Ac-PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT-resistant tobacco showed that the maximal tolerated N-Ac-PPT concentration would be less than 45 mM of the L-isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L-PPT content to toxic concentrations. In contrast, N-acetyl-L-phosphinothricyl-alanyl-alanine (N-Ac-L-PPTT), a substance also occurring during the biosynthesis of phosphinothricyl-alanyl-alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N-Ac-L-PPTT.

Production of male sterile transgenic plants

Ethylene controls many physiological and developmental processes in plants, including fruit and flower development, reproductive physiology, and responses to environmental stimuli. Ethylene exerts its effects through the ethylene receptor of plants. Ethylene receptor genes have been isolated in a variety of plant species, and in early studies, these genes were used to genetically engineer fruit ripening in tomato and flower senescence in petunia and carnation. Recently, we demonstrated that the over-expression of mutated melon ethylene receptor genes affected pollen development and induced a male sterile phenotype in transgenic plants. One major concern regarding genetically modified plants is the transgene flow through pollen dispersal, which may pose a potential impact to the environment, especially on genetic diversity. Therefore, the inducible male sterility system using mutated ethylene receptor genes could be a possible strategy for preventing pollen dispersal from these plants, thereby reducing the potential impact associated with transgenic plants. This review summarizes the studies on the inducible male sterility system that uses ethylene receptor genes.

Nuclear male sterility induced by pollen-specific expression of a ribonuclease

The promoter of a rice pollen-specific gene, PS1, has been fused to the Bacillus amyloliquefaciens Barnase gene which encodes a secreted ribonuclease. The PS1-Barnase chimeric gene has been introduced into tobacco. These transgenic tobacco plants show normal vegetative and floral development, but they display a range of reproductive properties from slightly reduced in fertility to completely sterile. Barnase mRNAs are detectable in the pollen from transgenic plants which do not show an obvious fertility-related phenotype, and in a few plants which have a mildly reduced-fertile phenotype. However, transgenic plants with a severely reduced-fertile or sterile phenotype do not accumulate detectable amounts of Barnase mRNA in their pollen, and the quality of their RNA is poor, presumably because of extensive RNA degradation. Reciprocal crosses between these transgenic plants and wild-type controls showed that the reduced-fertile phenotype is associated only with the transgenic pollen. When used as the female parent, these PS1-Barnase transgenic plants are fully fertile. Anthers in the severely sterile transgenic plants develop normally, but the majority of their pollen grains have abnormal morphology and they fail to germinate. These results indicate that expression of a pollen-specific cytotoxic gene induces lethality in pollen and may lead to severely reduced male fertility.

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.