Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Quantitative differences in sperm cells and organelles of tobacco (Nicotiana tabacum L.) grown under differing environmental conditions

In flowers grown at warm temperatures in environmental chambers and at cooler temperatures in the greenhouse, eight parameters of the sperm-cell organization of Nicotiana tabacum were examined during sperm cell maturation using serial ultrathin sectioning, transmission electron microscopy and quantitative cytology. Despite employing the same seed source, and similar soil and nutrient conditions, the surface area and volume of the cell, the nucleus and the chondriome were larger in flowers grown in growth chambers under warmer controlled conditions, whereas the number of plastids appeared to be the same, or slightly higher, in flowers grown under cooler greenhouse conditions. These results suggest that environmental conditions may influence the quantity of cytoplasmic organelles, including mitochondria and plastids, thus potentially influencing the likelihood of male cytoplasmic inheritance.     

Site-directed recombination in the genome of transgenic tobacco

Summary The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase 11 gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%–100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2–3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Localization of myosin on sperm-cell-associated membranes of tobacco (Nicotiana tabacum L.)

Summary Actomyosin interactions are reportedly the principal mechanism for the transport of nonmotile sperm cells of flowering plants inside the pollen tube and inside the embryo sac. Myosin has been demonstrated on the generative cell (the predecessor of sperm cells), although it is unclear from previous studies whether myosin is located directly on the plasma membrane of the male germ cells or on the external plasma membrane of the pollen cell that surrounds them. Immunogold scanning electron microscopy was used to localize myosin on isolated tobacco sperm cells, with and without associated membranes. When present, the pollen tube plasma membrane surrounding the sperm cells was labeled by an antimyosin antibody, as were pollen tube cytoplasmic organelles. Negligible labeling was observed directly on the plasma membrane of the sperm cells.     

Role of Ca2+ in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.)

Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to determine how different application rates of calcium may influence this metabolic process. Increased calcium in the nutrient solution in which tobacco plants were grown considerably reduced the foliar concentration of phenolic compounds. Calcium clearly exerted a positive influence on the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase) involved in the metabolism of the phenolics. High dosages of calcium (5 mM) promoted more oxidation than synthesis of these compounds, thus explaining the lower concentration of the phenolics.     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

In vitro induction of stigma-like and style-like structures from receptacle tissue in Nicotiana tabacum L.

Stigma-like and style-like structures were induced from the receptacle tissue in tobacco (Nicotiana tabacum L.). The presence of kinetin was necessary to induce these structures. The structures have a form similar to and the same function as normal stigmas and styles.     

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.     

Characterisation and immunolocation of an 87 kDa polypeptide associated with UDP-glucuronic acid decarboxylase activity from differentiating tobacco cells (Nicotiana tabacum L.)

UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown-Agarose. The native enzyme had an apparent M(r) of 220,000 which yielded a single subunit of 87,000 when analysed on SDS-PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around M(r) 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.     

Carbon metabolism enzymes and photosynthesis in transgenic tobacco (Nicotiana tabacum L.) having excess phytochrome

J.M. Keller et al. (1989, EMBO J. 8, 1005–1012) introduced a phytochrome gene controlled by a cauliflower mosaic virus 35S promoter into tobacco (Nicotiana tabacum L.) providing material to test whether several photosynthesis enzymes can be increased by one modification to the plant. We report here that this transgenic tobacco had greater amounts of all enzymes examined as well as greater amounts of total protein and chlorophyll per unit leaf area. Fructose bisphosphatase (E.C. 3.1.3.11), glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12), and sucrose-phosphate synthase (E.C. 2.4.1.14) were also higher when expressed per unit protein. However, ribulose-1,5-bisphosphate carboxylase (E.C. 4.1.1.39) amount per unit leaf protein was the same in transgenic and wild-type (WT) plants. Photosynthesis in the transgenic plants was lower than in WT at air levels of CO₂, but higher than in WT above 1000 bar CO₂. The photosynthesis results indicated a high resistance to CO₂ diffusion in the mesophyll of the transgenic plants. Examination of electron micrographs showed that chloroplasts in the transgenic plants were often cup-shaped, preventing close association between chloroplast and cell surface. Chloroplast cupping may have caused the increase in the mesophyll resistance to CO₂ diffusion. We conclude that it is possible to affect more than one enzyme with a single modification, but unexpected physical modifications worsened the photosynthetic performance of this plant.Abbreviations CABP 2-carboxyarabitinol 1,5-bisphosphate - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - GAP glyceraldehyde 3-phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase - SPS sucrose-phosphate synthase - WT wild type This research was supported by U.S. Department of Energy contracts DE-FG02-87ER60568 to T.D.S. and DE-FG02-88ER 13968 to R.D.V. We thank Drs. Joel Cherry and Howard P. Hershey for assistance with the transgenic plants.     

RNA,proteins and polyamines during gametophytic and androgenetic development of pollen in Nicotiana tabacum and Datura innoxia

Variations in polyamines, proteins and RNA during in vivo gametogenesis and in vitro androgenesis in Datura innoxia and in Nicotiana tabacum were studied. Spermidine was the major polyamine during gametogenesis in both species. Marked differences in proteins, RNA and polyamines were evident during meiosis and at the first haploid mitosis. In Nicotiana an unknown amine (X₆₀) appears at the beginning of the first haploid mitosis. At the same time a rapid increase in the concentrations of RNA and proteins is observed. In Datura, at the time of the first haploid mitosis there is large increase in amine and RNA levels followed by an arginine peak. During androgenesis, putrescine and spermidine were the major polyamines in both species. In Nicotiana during androgenesis an unknown amine (X₈₁) was observed together with putrescine and spermidine. This unknown compound peaks during the developmental stages of embryogenesis. In Datura androgenic induction was marked by an arginine peak followed by an increase in the putrescine and spermidine levels associated with maximum RNA. These biochemical events are tentatively correlated with structural changes during pollen development. The significance of these results is discussed in relation to the role of polyamines during gametogenesis and androgenesis.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.