The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Genomic factors controlling the lethality exhibited in the hybrid between Nicotiana suaveolens Lehm. and N. tabacum L.

Interspecific hybrid plants between Nicotiana suaveolens and N. tabacum exhibit lethal symptoms at the seedling stage and cannot grow to maturity. In this investigation, an attempt was made to clarify the genomic factors responsible for this lethality. N. suaveolens was crossed to N. sylvestris (genomic constitution: SS) and N. tomentosiformis (TT), these latter two species being the progenitors of N. tabacum (SSTT). From the cross N. suaveolens x N. tomentosiformis, many seedlings were obtained through ovule culture, and these subsequently grew to maturity without exhibiting any lethality. In the reciprocal crossing between N. sauvelons and N. sylvestris, only a few hybrid seedlings were obtained through ovlue culture and all died after unfolding their cotyledons when cultured at 28 °C. This lethality could be avoided by culturing the ovules at 36 °C. These features of hybrid lethality resembled those observed in the interspecific hybrid between N. suaveolens and N. tabacum. These findings suggest that the S genome in N. tabacum is responsible for the lethality exhibited in the hybrid between N. suaveolens and N. tabacum.     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

Somatic hybridization in Nicotiana: Segregation of organellar traits among hybrid and cybrid plants

Protoplasts from a nitrate reductase-deficient mutant of Nicotiana tabacum L. were fused with protoplasts from a stamen-less, cytoplasmically malesterile cultivar of tobacco containing the cytoplasm from N. suaveolens Lehm. Plants were regenerated from the fused protoplasts and characterized with respect to stamen development, chromosome number, and chloroplast composition. Of 29 regenerated plants, stamen production was restored in 26 plants and pollen production in 22. One plant was male sterile and two plants have never flowered. Analysis of the electrophoretic mobility of ribulose-1,5-bisphosphate carboxylase (RuBPcase) showed that 19 of the plants contained RuBPcase of the N. suaveolens type, six plants contained enzyme of the N. tabacum type, and four plants contained both types. Analysis of resistance to tentoxin in seedlings from 20 of the plants demonstrated that 14 had N. suaveolens-type chloroplasts, three had N. tabacum type, and three contained both types. Many of the plants which produced stamens and pollen still contained chloroplasts of the N. suaveolens type. Thus, the trait of cytoplasmic male sterility in tobacco is not an expression of the type of chloroplast genetic material.     

Osteophloeum platyspermum andVirola duckei (myristicaceae): newly reported as hallucinogens from Amazonian Ecuador

Quijos Quichua collaborators identifiedOsteophloeum platyspermum andVirola duckei (Myristicaceae) as sources of a psychoactive sap. This is the first reported hallucinogenic use of Myristicaceae from Ecuador. Species in Malpighiaceae and Solanaceae are more common sources of hallucinogens, but older Quichua still employ these myristicaceous species.Virola is used widely as a hallucinogen in other parts of Amazonia but there are no previous reports on the psychoactive use ofO. platyspermum. Field tests for the presence of alkaloids using Dragendorffs reagent were positive for both species. Like the Bora and Witoto in Peru, the Quijos Quichua consume myristicaceous hallucinogens orally. Most other indigenous peoples prepare psychoactive snuffs from the bark and sap.     

Hybrid lethality in interspecific hybrids between Nicotiana tabacum and N. suaveolens: evidence that the Q chromosome causes hybrid lethality based on Q-chromosome-specific DNA markers

Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens express lethality at 28°C. We carried out a cross between monosomic lines of N. tabacum lacking the Q chromosome and N. suaveolens by test-tube pollination and ovule culture at 28°C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36°C immediately after germination and cultured. We determined whether Q-chromosome-specific DNA markers were detected among hybrid seedlings. When hybrid seedlings cultured at 36°C were transferred to 28°C, hybrid seedlings in which Q-chromosome-specific DNA markers were detected expressed hybrid lethality, while hybrid seedlings in which Q-chromosome-specific DNA markers were not detected did not express hybrid lethality. From these results, we concluded that the presence of the Q chromosome of N. tabacum is related to hybrid lethality observed in crosses between N. tabacum and N. suaveolens. This is the first report that clearly demonstrates the relationship between a certain chromosome and hybrid lethality in the genus Nicotiana using chromosome-specific DNA markers. Additionally, we confirmed that the Q chromosome belongs to the S subgenome because Q-chromosome-specific DNA markers were detected only in N. sylvestris.     

Effect of essential oils from some higher plants against fungi causing damping-off disease

A screening of leaves of 25 taxa of angiosperms was made for their volatile toxicity against damping-off fungi. The volatile substances fromHyptis suaveolens andOcimum canum were toxic againstPythium aphanidermatum, P. debaryanum andRhizoctonia solani. The fungitoxicity of the leaves persisted for 15 d of storage. The volatile substances from the leaves ofO. canum were thermostable, while those fromH. suaveolens were thermolabile. The essential oils exhibited strong potency against the pathogens tested, non-phytotoxic nature to the host plants and superiority over commonly used synthetic fungicidesAgrosan G.N. andCaptan. The findings indicate the possibility to use these essential oils as potential natural fungicides in management of damping-off pathogens.     

Screening the receptorome for plant-based psychoactive compounds

Throughout time, humans have used psychoactive plants and plant-derived products for spiritual, therapeutic and recreational purposes. Furthermore, the investigation of psychoactive plants such as Cannabis sativa (marijuana), Nicotiana tabacum (tobacco) and analogues of psychoactive plant derivatives such as lysergic acid diethylamide (LSD) have provided insight into our understanding of neurochemical processes and diseases of the CNS. Currently, many of these compounds are being used to treat a variety of diseases, such as depression and anxiety in the case of Piper methysticum Kava Kava (Martin et al., 2002; Singh and Singh, 2002). G-protein coupled receptors (GPCRs) are the most common molecular target for both psychoactive drugs and pharmaceuticals. The "receptorome" (that portion of the genome encoding ligand reception) encompasses more than 8% of the human genome (Roth et al., 2004) and as such provides a large number of possible targets for psychoactive drug interactions. A systematic, comprehensive study is necessary to identify novel active psychoactive plant-based compounds and the molecular targets of known compounds. Herein we describe the development of a high throughput system (HTS) to screen psychoactive compounds against the receptorome and present two examples (Salvia divinorum, the "magic mint" hallucinogen and Banisteriopsis caapi, the main component of Ayahuasca, a psychoactive beverage) where HTS enabled the identification of the molecular target of each compound.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Inheritance behavior of information coding for small subunit polypeptides of fraction 1 protein

In various genera of plants, the small subunit of fraction 1 protein is often composed of more than one kind of polypeptide; these differ in isoelectric points and amino acid composition. Previous analysis of numerous individual progeny of Nicotiana tabacum (two kinds of polypeptides), N. glauca + N. langsdorffii parasexual hybrids (three kinds), and other examples showed no change in F-1 protein composition as a consequence of alternation of generations. Experiments reported here show that absence of one member of each of the 24 different pairs of chromosomes in an N. tabacum monosomic series and also absence of the “S” pair in a nullisome did not affect F-1 protein composition. Absence of the “E” pair caused reduction in the amount of the least acidic of the two kinds of N. tabacum small subunit polypeptides. The question of how many individual progeny of self-fertile hybrids would have to be analyzed to detect segregation of genes coding for F-1 protein small subunit polypeptides, if segregation occurs, was answered by analysis of F₁ hybrids between N. otophora and N. tomentosiformis, and two subspecies of N. suaveolens, together with their F₂ progeny. In both cases, analysis of 16 progeny was sufficient to demonstrate a segregation pattern of two F₁ hybrid type to one each of the two parental types. Therefore, in the absence of segregation, it is likely that coding information for different kinds of F-1 protein small subunit polypeptides is sequestered on heterologous chromosomes, as postulated in previous reports.     

Stylar water potential and unilateral interspecific incompatibility inSolanaceae

Water and osmotic potentials were measured in young and mature styles of selfcompatible (SC) and selfincompatible (SI) species ofSolanaceae. For mature flowers, the stylar water and osmotic potentials were considerably lower in SI speciesNicotiana alata andPetunia hybrida than in SC speciesN. sylvestris andN. tabacum. Stylar water content (in % fresh mass) was also significantly lower in SI species (N. alata, P. hybrida, diploid clones of potato) than in SC species (N. acuminata, N. glauca, N. paniculata, N. sylvestris, N. tabacum). For flower buds younger than 2 d before anthesis, no differences in stylar water potential betweenN. alata (SI) andN. tabacum (SC) were observed. During further flower bud development the stylar water potential inN. alata decreased more expressively than inN. tabacum. Pollen culture in media with various concentrations of sucrose or polyethylene glycol revealed that optimal water potential for pollen tube growth was lower inN. alata than inN. tabacum. The differences were similar as for differences in stylar water potential. The results are considered in relation to possible involvement of stylar water potential in unilateral interspecific incompatibility inSolanaceae.     

Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana

Summary Variation in mitochondrial protein synthesis and genome organization was investigated. Three different alloplasmic cytoplasmic male-sterile Nicotiana tabacum cultivars, carrying N. repanda, N. suaveolens or N. debneyi cytoplasm, were analysed together with corresponding male-fertile parental and restored material. Although several differences were detected in the proteins synthesized by isolated mitochondria from the male-sterile and male-fertile plants, most of these were related to the origin of the mitochondria. However, a 23 kD protein was synthesized in the male-sterile cultivar carrying N. debneyi mitochondria, but not in other lines containing this cytoplasm. This protein was also present in the male-fertile parent containing N. tabacum mitochondria. Only the enhanced production of a 30 kD protein in the lines carrying mitochondria from N. repanda or N. debneyi was exclusively correlated with CMS. This protein was not present in any of the corresponding male-fertile parental and restored lines. Restriction enzyme analysis of mitochondrial DNA revealed a difference in abundance of a 5.6 kb XhoI fragment between lines containing N. debneyi mitochondria. No rearrangements of mitochondrial DNA was found between male-fertile and male-sterile lines carrying N. repanda or N. suaveolens cytoplasm. These results might indicate that CMS in alloplasmic Nicotiana cultivars is caused by alterations in the expression of mitochondrial genes, rather than by induced changes in the genome.     

Qualitative microbiological changes during decomposition of plant material in a sandy sierozem soil

The successive qualitative microbial changes during the decomposition of bajra stalk in a sandy sierozem soil were studied.Alternaria spp.,Aspergillus spp.,Cladosporium spp.,Fusarium spp.Gliocladium spp.,Mucor spp. andRhizopus spp. were most common fungi. The bacteria observed wereAchromobacter, Arthrobacter, Bacillus, Micrococcus, Pseudomonas andXanthomonas. Cellvibrio andCellulomonas were also observed.     

Autoradiographic screening for potential heterotrophs in natural algal populations of Lake Kinneret

Microautoradiography was used to screen natural phytoplankton populations from Lake Kinneret for their ability to take up radioactive organic substrates (glucose, acetic acid, amino acids, and glycollic acid. Several of the important Chlorophyta (Pediastrum spp.,Actinastrum hantzschii, Kirchncriella spp.,Coelastrum spp.,Scenedesmus spp., andTetraëdron spp.) and Cyanophyta (Microcystis spp.,Merismopedia sp.,Chroococcus spp., andAnabaena spp.) showed consistent organic uptake. However, the principal bloom former, the dinoflagellatePeridinium cinctum fawestii and most of the pelagic algae observed, never assimilated any of the above substrates. Autoradiographic surveys permit rapid screening of algal populations for species which are capable of taking up organic matter and can indicate potential facultative heterotrophs.     

Effect of X-radiation on pollen tube growth and seed setting in crosses between Nicotiana tabacum and N. rustica

Summary In vitro andin vivo studies of the effect of irradiating pollen with different doses of X-rays were carried out inNicotiana rustica andN. tabacum. Dosages upto 9600 r were found to enhance the rate of growth of pollen tubers per unit time. Advantage was taken of this observation to overcome the incompatibility normally found in the crossN. tabacum x N. rustica as a result of the inadequate growth ofrustica pollen tubes intabacum style. Thus, whenrustica pollen exposed to 4800 r and 9600 r of X-rays were used to pollinatetabacum, fertilization resulting in the formation of viable hybrid seeds occurred. In the crossN. rustica×N. tabacum there is partial seed failure as a result of the hyperplastic development of the nucellar and endothelial cells. However, when X-rayedtabacum pollen were used in the cross, seed development registered a marked improvement. Since there is now evidence for suggesting that the contents of even those pollen tubes which do not participate in fertilization have some role to play in the development of the seed, it seems likely that radiation-induced biochemical changes in pollen may prove advantageous when conditions for seed development are abnormal as in instances of somatoplastic sterility. Besides such indirect biochemical effects, irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals. Thus, there is much scope for investigating the use of radiation to overcome inter-specific and inter-generic cross-incompatibility barriers.With 2 Figures in the Text     

Nuclear DNA,heterochromatin and phylogeny of Nicotiana amphidiploids

There are significant differences in nuclear DNA amount between both diploid and amphidiploid species of Nicotiana. Owing to the higher DNA density in the interphase nuclei of the amphidiploids DNA amounts tend to be underestimated by microdensitometry. After applying necessary corrections to amphidiploid readings it was found that: (1) The nuclear DNA amount in the tetraploid N. rustica is not significantly different from the sum of nuclear DNA amounts in reputed diploid parents, N. undulata and N. paniculata. (2) It is well established that N. sylvestris is one of the diploid progenitors of N. tabacum. The sum of the nuclear DNA amounts in N. sylvestris and N. tomentosiformis is not significantly different from that of the amphidiploid N. tabacum. In contrast the sum of the DNA amounts in N. sylvestris and N. otophora is significantly higher than that in N. tabacum. Observations and measurements of the amount and distribution of heterochromatin in interphase nuclei of the diploid and tetraploid species give further support to the conclusion that N. tomentosiformis rather than N. otophora is the second diploid progenitor of N. tabacum.     

Cytoplasmic male sterility in Nicotiana, restoration of fertility, and the nucleolus

Plants with the cytoplasm of Nicotiana repanda and the chromosomes of N. tabacum produce nonfunctional and feminized anthers. Introduction of a satellited fragment chromosome, apparently derived from N. repanda, restores normal anthers and pollen fertility. The fragment is somatically stable and addition-homozygotes transmit it to the great majority of their offspring. Cells with the fragment exhibit amphiplasty, i.e. , in plants having one or two fragments, nucleolar organizers of N. tabacum are suppressed and the nucleoli are entirely or largely produced by the fragments. Formation of nucleoli by organizers from N. repanda in N. repanda cytoplasm may thus be a condition for male fertility. The manner in which nucleoli may influence the development of originally indeterminate sex primordia is discussed. Further cytological studies of cytoplasmically male-sterile plants and restorer chromosomes are needed to decide whether the observed relationship is of functional significance or merely accidental.     

The Transmitting Tissue in Brugmansia suaveolens L.: Ultrastructure of the Stylar Transmitting Tissue

The stylar transmitting tissue of the angel's trumpet, Brugmansia(Datura) suaveolens, was studied at two developmental stages:about 6 d before anthesis and after anthesis. Histochemicallocalization of polysaccharides was carried out with PATAg andimmunohistochemistry with gold-conjugated antibodies recognizingpectins. Before anthesis the transmitting tissue forms a centralcore of polyhedral meristematic, still dividing, cells withnarrow intercellular spaces. Epitopes for unesterified pectinsare present in the walls and the spaces between the cells, whilemethylesterified pectins are confined to the middle lamellaand intercellular spaces. PATAg positive material and the antibodyagainst unesterified pectin was found in plasmalemma invaginationsand multivesicular bodies. Dictyosome cisternae and vesiclescontained epitopes for both kinds of pectins. Plastids are poorlydifferentiated and lack starch. Nutrients are stored as lipidbodies, which are digested by small vacuoles. After anthesisthe transmitting tract cells form cylindrical files separatedby voluminous spaces filled with a mucous secretion reactingwith PATAg and with the antibody against unesterified pectins.Dictyosome vesicles contain epitopes for the same kind of pectins.The cells are vacuolized and have leucoplasts. This study showspectin synthesis by different parts of the endomembrane systemand changes in pectin esterification during stylar development.Copyright1993, 1999 Academic Press Brugmansia suaveolens, immunocytochemistry, pectin, secretion, style, transmitting tissue     

TUMORS OF INTERSPECIFIC NICOTIANA HYBRIDS. I. EFFECT OF TEMPERATURE AND PHOTOPERIOD UPON FLOWERING AND TUMOR FORMATION

To establish the flowering and tumor-forming response of the hybrid Nicotiana suaveolens Lehm. × Nicotiana langsdorffii Weinm., it was grown with the parental species under two temperatures and night-length photoperiods. All plants flowered earlier with a 2-hr night interruption. The floral response of N. suaveolens was more sensitive to temperature than that of N. langsdorffii. The response of the hybrid was similar to N. suaveolens in the warm temperatures (24–27 C) and similar to N. langsdorffii in the cool (15–21 C). Tumors on roots of the hybrids were much more pronounced on plants grown in higher temperatures.     

Genes in S and T subgenomes are responsible for hybrid lethality in interspecific hybrids between Nicotiana tabacum and Nicotiana occidentalis

Background

Many species of Nicotiana section Suaveolentes produce inviable F₁ hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.

Methodology/Principal Findings

Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.

Conclusions/Significance

Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis.