Swords into plowshares? Nicotiana sylvestris does not use nicotine as a nitrogen source under nitrogen-limited growth

Whether or not a plant can recover its investment of resources in a chemical defense is central to the mobile-immobile metabolite dichotomy of the resource availability theory. Biochemical measures of metabolite turnover have been used to estimate this trait, but they do not address the ecological question of resource recovery. Numerous studies have found that many Nicotiana species, which normally produce the nitrogen-intensive defense metabolite, nicotine, can rapidly take up and metabolize exogenously administered nicotine from hydroponic solutions. However, Baldwin et al. (1994) found no evidence for turnover of endogenously produced nicotine in pulse-chase experiments using ¹⁵NO₃ as the biosynthetic precursor in N. sylvestris. Given that the capacity to metabolize nicotine exists, we asked (1) whether N. sylvestris could metabolize exogenously fed nicotine and sustain growth under nitrogen-limited conditions and (2) whether leaf damage alters the plants' ability to use nicotine as a nitrogen source. We fed plants with sufficient nicotine in hydroponic culture to increase their nitrogen pools by 70% at the time of nicotine feeding; in 6–10 consecutive harvests over 28–35 days, we measured the biomass of roots, leaves and stems, and the total nitrogen pools of these plant parts as well as the pools of nicotine, nornicotine and myosmine of these plant parts in undamaged nicotinefed and control plants and finally, in a separate experiment, in nicotine-fed damaged and undamaged plants. Nicotine feeding increased nicotine pools by 1.2 times, which was not sufficient to significantly increase total nitrogen pools at the end of the experiment. Nicotine-fed plants rapidly demethylated their acquired nicotine pools to nornicotine, but did not process the alkaloid pool further than myosmine over the duration of the experiment. Leaf damage significantly increased the nicotine pool, but did not significantly alter the processing of the exogenously acquired nicotine. We conclude that N. sylvestris does not recover the nitrogen invested in nicotine even under nitrogen-limited growth, that the rapid metabolism of exogenously introduced nicotine is likely a detoxification pathway, and that these plants are homeostatic with regard to their nicotine pools.     

Constraints on an induced defense: the role of leaf area

Folivory results in both leaf damage and the loss of photosynthetic capacity. Leaf damage activates the production of induced defenses, but diminished photosynthetic capacity resulting from lost leaf area may impair a plant's ability to respond defensively. Because damage-induced nicotine production in Nicotiana sylvestris (Solanaceae) is an energy-demanding, active process, we predicted that the loss of leaf area would constrain this plant's ability to produce an induced nicotine pool. We examined our prediction in an experiment which combined leaf puncture and removal protocols, quantified induced nicotine pools on a whole-plant basis, and accounted for losses in the nicotine pool due to removed leaves and lost growth potential. In contrast to our prediction, leaf removal did little to diminish the growth-corrected estimates of the induced nicotine pool in plants with sufficient damage cue; only when plants had lost 88% or more of their leaf area did the induced nicotine pool decline significantly. These results demonstrate that the induced defense is relatively insensitive to current photosynthetic capacity. In contrast to the size of the induced nicotine pool, the concentration of nicotine in the remaining shoot tissues continued to rise as puncture damage increased over all defoliation levels tested. The mechanisms responsible for inducible nicotine production may have evolved as a means of providing shoot tissues with protection that is proportional to the amount of damage incurred while keeping production costs constant for the remaining plant parts.     

Association of diamine oxidase and <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine hydrolase in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> extracts

The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80 of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Somatic hybrid plants of Nicotiana glauca and Nicotiana tabacum obtained by protoplast fusion

Somatic hybrid plants were produced by fusion of protoplasts from cell cultures of the Nicotiana tabacum L. sulfur mutant Su/Su and from leaf mesophyll of Nicotiana glauca Graham. After fusion the N. glauca protoplasts failed to survive under the selected culture condition. From the hybrid cells light green shoots were produced. The hybrid plants exhibited intermediate characters between parental species with respect to leaf morphology, trichome density, floral structure and flower color. The chromosome number of 25 hybrid plants was 2n = 72 and both N. glauca and N. tabacum chromosomes were identified in the hybrids. Results of isoenzyme analysis showed bands of both parents and a specific (hybrid) band for aspartate amino-transferase. Small subunit fraction-1-protein of somatic hybrids also consisted of the sum of N. glauca and N. tabacum bands. Leaf spot formation associated with the Su locus of N. tabacum was observed in somatic hybrids.     

Effects of soil water on photosynthetic characteristics and leaf traits of Cyclobalanopsis glauca seedlings growing under nutrient-rich and -poor soil

Cyclobalanopsis glauca is a dominant species in mid-subtropical forest, and usually plays an important role in forest ecosystems. However, it often suffers redundant precipitation or water stress, which often concurs with high temperature, nutrient depletion and strong irradiance. The study presented in the paper hypothesized that soil water exerted strong influence on leaf gas exchange and traits. The objective of this study was to clarify the effect of soil water changes on photosynthetic characteristics and leaf traits and their relationships of C. glauca seedlings growing on nutrient-rich and nutrient-poor soil at three water levels. The study measured the specific leaf area (SLA), nitrogen content, chlorophyll concentrations and photosynthetic light response curve. Its results showed that there were no differences in leaf size, leaf dry weight, SLA, leaf dry matter content, Leaf nitrogen concentration and Leaf chlorophyll between the two soil nutrient treatments, while these parameters differed significantly among different water levels for either of the treatments. There were large variations in leaf photosynthetic parameters and leaf traits among the different water treatments, indicating different response patterns of C. glauca seedling and its adaptation to the different soil water conditions. There were no significant differences in light-saturated photosynthetic rate (A_max) and apparent quantum yield (Ø) between the nutrient-rich and nutrient-poor soils, which indicated that the C. glauca seedlings could maintain similar capacities in different soils that differed in nutrient condition. As to the relation between the photosynthesis and leaf traits, the A_max and PNUE were positively correlated with the SLA, respectively, but the SLA had significant negative relationship with the leaf N (P<0.01) in nutrient-rich soil. In contrast, both A_max and PNUE were significantly negatively correlated with the SLA, respectively (P<0.01); and the SLA was not significantly positively correlated with the leaf nitrogen concentration of the nutrient-poor soil (P>0.05). The specific leaf areas (SLA), nitrogen and chlorophyll concentrations as well as other photosynthetic features were influenced in a coordinative manner by the soil water. The relation among the A_max, PNUE and the N_mass, SLA could be described as a binomial equation and a liner negative regression for the nutrient-rich and nutrient-poor soil, respectively. In conclusion, soil water was more constraining factor than the soil nutrient to the photosynthesis of C. glauca seedlings, nutrient-rich soil could offset some negative influence resulting from soil water deficit on LSP and LCP. Factors affecting the variations of photosynthetic characteristics and leaf traits of C. glauca seedlings differed between the nutrient-rich and nutrient-poor soils.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

The PELPIII glycoproteins in Solanaceae: stylar expression and transfer into pollen tube walls

The class III pistil-specific extensin-like proteins (PELPIII) of Nicotiana tabacum accumulate in the intercellular matrix (IM) of the style transmitting tissue (TT). After pollination, the 110–140 kDa PELPIII is translocated from the IM into the pollen tube walls. PELPIII-like sequences have been found in several solanaceous species. These sequences are expressed in mature non-pollinated styles at both RNA and protein level. Of the genus Nicotiana, the species N. alata, N. x sanderae and N. sylvestris (section Alatae), and N. tomentosiformis and N. otophora (section Tomentosae) showed an expression level of PELPIII homologues similar to that in mature styles of N. tabacum. PELPIII genes were absent in the most ancient species studied, namely N. trigonophylla (section Trigonophyllae). To study the species dependence of the translocation of PELPIII into the pollen tube wall in tobacco, interspecific pollinations on N. tabacum pistils were carried out with pollen from the incongruous species N. rustica, N. trigonophylla and Petunia hybrida, where PELPIII homologues are absent in the style. Immunocytological tests showed that the N. tabacum PELPIII is translocated into the pollen tube walls of all three species. Thus, the pollen tube walls of these species do not form a barrier for IM compounds such as the 110–140 kDa PELPIII and the absence of any possible effect of PELPIII on pollen tube growth cannot be due to failure of PELPIII transport through the wall. The importance of these findings is discussed with respect to the evolutionary origin of PELPIII, the pollen pistil interaction, the function of style TT-specific proteins and the physical properties of pollen tube walls.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

Serological relationships of Fraction I proteins from species in the genusNicotiana

The serological relationships of Fraction I proteins from 62 species of the genusNicotiana have been determined by qualitative double diffusion in agar gels, using antisera raised against crystalline Fraction I proteins from four species,N. glauca, N. glutinosa, N. gossei, andN. tabacum. The relationships obtained are, in most cases, similar to the relationships of the species based on morphological and cytogenetic criteria. In general, Fraction I proteins from species within the same section are serologically identical. However, serological examination of Fraction I proteins draws attention to certain species, for example,N. glauca, N. sylvestris, andN. acaulis, whose classification by traditional methods has been difficult.     

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumors of N. glauca x N. langsdorffii

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.     

Construction of phylogenetic tree forNicotiana species based on RAPD markers

To apply random amplified polymorphic DNA for analysis of phylogenetic relationships, we used 34 synthetic oligonucleotides as primers to examine interspecific and intraspecific variations among 18 genotypes, nine species ofNicotiana. The nine species used in this study belong to sectionsTomentosae andAlatae. In addition, we attempted to clarify the taxonomic position ofN. sylvestris. A total of 354 distinct DNA fragments were obtained by polymerase chain reaction. Pair-wise comparisons of unique and shared amplification products were used to generate Jaccard's similarity coefficients and Nei and Li's similarity coefficients with the computer software of numerical taxonomy and multivariate analysis system. On the basis of the dendrogram constructed with the similarity coefficients, the 18Nicotiana genotypes were divided into two clusters. The classification analyzed by RAPD markers is in accordance with the classification of Goodspeed thatN. sylvestris is a member of sectionAlatae.     

Transmission genetics of the somatic hybridization process in Nicotiana

Summary Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P ^– genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Effects of in vitro tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio

The levels of two subunits of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), total soluble proteins, carbon and leaf nitrogen content, and photosynthetic pigments in various plants (avocado, oak, olive, and strawberry) grown in vitro and ex vitro were analysed. Compared to ex vitro grown plants, micropropagated avocado, oak, and strawberry showed a markable decrease in large subunit Rubisco. However, the small subunit only decreased in strawberry and oak. Contrary to this, olive did not reveal any difference in the level of either subunit. The C/N ratio increased significantly in in vitro grown plants, except in the case of olive, where an opposite behaviour was found. Leaf chlorophyll concentration on unit mass basis was higher in all the in vitro plants than in those of greenhouse- grown plants. Only avocado plantlets showed a statistically significant decrease in total soluble proteins. Further, overall data suggest that in vitro cultural conditions have a species-specific influence on large and small subunits of Rubisco, independent of the protein, chlorophyll, or nitrogen level.     

Transgenic tobacco plants expressing a rice cysteine synthase gene are tolerant to toxic levels of cadmium

Plants tolerate heavy metals through sequestration with cysteine-rich peptides, phytochelatins. In this reaction, the rate limiting step is considered to be the supply of cysteine, which is synthesized by cysteine synthase (CS, EC 4.2.99.8) from hydrogen sulfide andO-acetylserine. In this study, we transformed tobacco (Nicotiana tabacum) plants withRCS1, a cytosolic cysteine synthase gene of rice (Oryza sativa), and examined their sensitivity to cadmium. The transgenic plants had up to 3-fold higher activity of cysteine synthase than wild-type plants. Upon exposure to cadmium, they exhibited obvious tolerance with much greater growth than wild-type plants. The level of phytochelatins in shoots was higher in transgenic than in wild-type plants after cadmium treatment, suggesting that cadmium was actively trapped by phytochelatins. However, the cadmium concentration per g fresh weight of whole transgenic plants was 20 percnt; lower than that of wild-type plants, suggesting cadmium to be either actively excreted or diluted by fast growth. Genetic analysis of progenies clearly showed segregation of cadmium tolerance, indicating that the trait resulted from the introduced gene. These results suggest that introduction of a cysteine synthase gene into tobacco plants resulted not only in high level production of sulfur-containing compounds that detoxify cadmium, but also in active elimination of cadmium toxicity from plant bodies.     

Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance

Summary A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco cybrids and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.