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Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species. [ABSTRACT FROM AUTHOR]

Floral traits are used as signals to attract pollinators and play an important role in species identification and isolation. Nicotiana is a genus with a diverse range of flower morphologies, colours, and pollination systems related to a natural history of hybridization, a driver of speciation in this genus. Nicotiana alata and Nicotiana forgetiana are pollinated by hawkmoths and hummingbirds, respectively, and a putative hybrid population was recently found. This population presents flowers with intermediate phenotypes providing an opportunity to investigate the impact of hybridization on floral trait variation. Here we investigated the floral shape, floral pigments, and nectar traits in a putative hybrid population, hereafter termed atypical populations (AP) compared to phenotypes of allopatric populations of both species. We found a high variation in floral pigmentation in AP plants and observed phenotype segregation in some genotypes. Nicotiana forgetiana and AP plants showed the same flavanol peaks as Nicotiana alata , suggesting the same UV-absorbent phenotype attractive to hawkmoths. The geometric morphometric results showed that the intermediate-coloured flowers had similar floral shape and size to N. alata. Our results suggest that the putative hybrid population would be able to attract the same parental pollinator—hawkmoths and hummingbirds—making backcrossing events possible and attracting other pollinators, such as bees. [ABSTRACT FROM AUTHOR]

Tetranychus evansi Baker & Pritchard, 1960 (Tetranychidae) is a phytophagous mite with wide distribution and high importance for agriculture. This study reports the occurrence of T. evansi in Nicotiana alata Link & Otto, Nicotiana forgetiana Sander ex W. Watson (Solanaceae) and their putative hybrids in a greenhouse in Rio Grande do Sul state, Brazil. Different stages of development of T. evansi were found in N. alata and N. forgetiana and in their putative hybrids.

Floral shape and size play a role in plant diversification and reproductive isolation. Different floral forms can rise by selective pressures by pollinators/ecological constraints and/or genetic drift. Nicotiana (Solanaceae) has c. 82 currently recognized species grouped in 13 sections. Nicotiana forgetiana and N. alata belong to section Alatae and present different floral traits related to their primary pollinators. These species co-occur in a region of plant endemism in southern Brazil marked by a mosaic between open grasslands and Araucaria forest. Here, we conducted a population-level sampling across the range of N. forgetiana and combined geometric morphometric analyses and ecological niche modelling to shed light on the patterns underlying intraspecific floral shape variation. Corolla shape and size varied significantly across the geographical distribution of N. forgetiana and ' rastroensis ', a putative new species. These floral shape differences were related to variations in temperature, precipitation and elevation. We also observed a range of intermediate floral traits in two populations, and our results of geometric morphometric analyses reveal morphological overlap between N. alata and N. forgetiana. Moreover, we found that habitat suitability for both species was impacted by past climatic oscillations, with severe reduction for N. forgetiana during the Last Glacial Maximum. We discuss the results to shed light on the evolution of N. forgetiana. [ABSTRACT FROM AUTHOR]

Usage of different xenobiotics, in particular ionizing radiation, for the modification of growth properties of pollen tubes is widely spread due to the possibility to apply gamma-irradiated pollen for mutagenesis and apomixis induction as well as transgenosis implementation. In view of this, studying gamma-irradiated pollen germination and growth in vitro would be helpful not only for the determination of ionizing radiation effects on pollen but also elucidation the special features acquired by pollen through gamma-irradiation in wide dose range. The research aimed at analysis of dynamics and kinetics of Nicotiana alata pollen tubes formation and further growth on the artificial nutritional medium consisted of 10% sucrose and 0,01% boric acid in sterile tap water at +28°C and luminescent illumination of 800 lx. Pollen was collected before anthesis and irradiated in dry state by gamma-rays in the dose range 5 3000 Gy. Application of pollen drop culturing made possible us to follow the dynamics of pollen germination and pollen tube growth in vitro. Thus, unirradiated control pollen was shown to germinate rapidly within three hours after sowing on the water solution of nutrients, the latent period before germination being equal to half an hour. Gamma-irradiated by 5 Gy Nicotiana alata pollen proved to be of the same germination qualities as control. Meanwhile gamma-irradiation of pollen by 300 Gy caused the increase of latent period before pollen germination up to 1,2 hr, the whole germination term reaching 7 hr. Simultaneously the percentage of germinated pollen decreased to 62% comparing with control where pollen germination was equal to 82%. The further radiation dose elevation resulted in gradual elongation of latent period before pollen germination up to 2,8 hr at the dose of 800 Gy and 3,8 hr at the dose of 1500 Gy. Apart from this, pollen gamma-irradiation stipulated the slowing of pollen tube formation, the latter being equal to 5,2; 2,5 and 0,7 pollen-tubes/hr at 800 Gy, 1200 Gy and 1500 Gy, respectively. Analysis of pollen-tube growth dynamics has elucidated that in control this process proceeded in five steps. At stage 1 pollen-tubes immediately after germination commenced to grow rapidly during 2,5 hr. Afterwards pollentube growth slowed down. By 8th hr of pollen cultivation in vitro the second raise of pollen-tube growth velocity appeared which continued within three hours and resulted in evident elongation of pollen tubes up to 92% of their final mean. At the fourth stage pollen-tubes reached their final length and entered the termination period (stage 5). Gamma-irradiation of tobacco pollen by 5 Gy did not change the shape of pollen-tube growth curve. However, dose increase to 300 Gy resulted in decrease of stage 3 duration associated with second raise of pollen-tube growth velocity. Further radiation dose elevation sequentially to 800 Gy, 1200 Gy and 1500 Gy caused full elimination of stage 3. Moreover, pollen-tube growth at high doses 1200 Gy and 1500 Gy proceeded with the same speed without any signs of its acceleration or slowing. Absence of second enhancement of pollen-tube growth at high radiation doses gives insight in mentor properties of gamma-irradiated pollen which promotes incompatible pollen growth in pistil and let it invade embryo-sacs in first turn. In addition, its worth mentioning that high radiation doses caused pollen tips rupturing and issuing of pollen cytoplasm outwards.

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Summary: In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxhSS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155‐Cys185 disulphide bond of SC10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection. Significance Statement: S‐RNase is very well known for its role in S‐RNase‐based self‐incompatibility and in some types of interspecific pollen rejection. Through a dominant‐negative approach, we show that a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, is required for both types of pollen rejection. This is a significant advance, although what sets our new contribution apart is that we show that NaTrxh reduces an S‐RNase disulphide and increases its activity, providing a remarkable level of insight into the biochemical mechanism of S‐RNase‐dependent pollen rejection. [ABSTRACT FROM AUTHOR]

The S -specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner , but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S -specific pollen rejection in Nicotiana. • NaStEP specifically and irreversibly inhibits a subtilisin-like protease. • A subtilisin-like processes the native NaStEP into two lower molecular weight peptides. • NaStEP also blocks the current of the voltage-dependent potassium channel Kv 1.3 when expressed in Xenopus oocytes. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase ( NalaPDS ) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species., Competing Interests: Authors QG and JDZ were employed by the company China Tobacco Yunnan Industrial Co. LTD,. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor LY declared a past co-authorship with the author XS. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Yuan, Zeng, Liu, Yu, Tong, Zhang, Gao, Wang, Sui, Xiao and Huang.)

In Solanaceae, self-incompatibility is a genetic mechanism that prevents endogamy in plant populations. Expression of the S- determinants, S-RNase, and SLF , is tightly regulated during pistil and pollen development. However, the molecular mechanism of gene expression regulation in S-RNase-based self-incompatibility systems must be better understood. Here, we identified a 1.3 Kbp sequence upstream to the coding region of the functional S C10 -RNase allele from the self-incompatible Nicotiana alata , which directs S C10 -RNase expression in mature pistils. This S C10 -RNase promoter includes a 300 bp region with minimal elements that sustain the S C10 -RNase expression. Likewise, a fragment of a transposable element from the Gypsy family of retrotransposons is also present at the −320 bp position. Nevertheless, its presence does not affect the expression of the S C10 -RNase in mature pistils. Additionally, we determined that the S C10 -RNase promoter undergoes different DNA methylation states during pistil development, being the mCHH methylation context the most frequent close to the transcription start site at pistil maturity. We hypothesized that the Gypsy element at the S C10 -RNase promoter might contribute to the DNA methylation remodeling on the three sequence contexts analyzed here. We propose that mCHH methylation enrichment and other regulatory elements in the S-RNase coding region regulate the specific and abundant S C10 -RNase expression in mature pistils in N. alata. • A 320 bp minimal S C10 -RNase promoter directs expression in mature pistils. • A transposable element in the S C10 -RNase promoter does not affect gene expression. • The promoter undergoes different DNA methylation states during pistil development. • The mCHH methylation context is the most remarkable in mature pistil. [ABSTRACT FROM AUTHOR]

Gamma-irradiated pollen is very often used as a tool for genetic reconstruction of plant cell. In view of this the necessity of pursuing detailed research in order to develop a basic radiobiologic theory for the explanation of discovered phenomena has emerged. The present research aimed at studying gamma-irradiation effects of different doses on germination of tobacco (Nicotiana alata) pollen on artificial feeding medium, growth properties of pollen tubes and mitotic division of generative cell on sperms. The dry tobacco pollen was gammairradiated in the dose range 50 - 3000 Gy and then germinated in the solution of 10% sucrose and 0,01% boric acid in sterile tap water. Germination and the length of pollen- tubes was determined by the aid of ocular-micrometer under the light microspore "MBU - 3" at the magnification ×150. The pollen nuclei were stained with 2% acetoorsein. Gamma-radiation was shown to gradually suppress germination and pollen tube growth, the doses higher than 2000 Gy being critical for said events. Meanwhile the generative cell division on sperms completely ceased at the dose 300 Gy and higher ones. Simultaneously the dose-dependent slowing of pollen tube grown speed was established. The multiplicity of physiological changes which emerged in pollen after gamma-irradiation, to our opinion, was in favour of gynogenesis and apomixis occurrence. [ABSTRACT FROM AUTHOR]

Nicotiana alata has long been a model species for studying gametophytic self-incompatibility (SI) in Solanaceae. Genetically based SI systems are widespread in flowering plants and function to reduce homozygosity and avoid inbreeding depression. However, out-crossing is always unfulfillable for populations with few S-alleles, such as in cultivated clonal populations or populations founded by few individuals. Under such conditions, there is a pressing demand to self-reproduce and pass on genetic information. We carried out a series of pollination and pollen tube observation experiments to explore how homozygous self-incompatible N. alata (S-allele identified and S-RNase active) sets fruits and to explore pseudo-self-fertility in these individuals. We observed several kinds of pollen tube abnormalities, including swelling, furcation, curve and inflexion, tip bursting and irregular callose plug deposition for flowers pollinated at different developmental stages. In addition, SI strength in this line of N. alata plants changed with floral age and flowering stages. That is, flowers became more self-compatible with floral aging, and SI strength in flowers at the end of flowering stages (stage III) was lower and more fruits were set at the top of the pseudo-raceme. Finally, SI strength also varied between individual flowers at the same plant and development stages. However, SI strength varied insignificantly among cultivated N. alata plants homozygous for the same S-allele. These findings indicate that SI in N. alata is plastic, with fruits set by aging flowers, especially flowers at stage III. [ABSTRACT FROM AUTHOR]

Haploid plants of Nicotiana alata were cultured in vitro on MS medium with IAA + KIN. The resulting plantlets were irradiated using gamma radiation doses of 10, 15, 20 and 25 Gy. Single node pieces were cut and transferred onto fresh MS medium. Gamma radiation doses caused the death of 9% and up to 28% of explants. NaCl concentrations caused the death of 8% up to 36% of explants, while the combined effect between gamma radiation doses and salinity had an impact suffused on the percentage of survival. The combined effect of gamma radiation doses 20 Gy and 25 Gy on NaCl concentrations of 100, 150 and 200 mM were deadly. Even more, the combined effect of gamma radiation doses and salinity had a severe negative impact on both the proline content and total soluble protein. Random amplified polymorphic DNA (RAPD) analysis was used to determine the degree of genetic variation in treated haploid Nicotiana alata plants. Total genomic DNAs from different haploid plantlets treated were amplified using five arbitrary primers. Two hundred and seventy bands were detected from plantlets irradiated with doses of 15, 20 and 25 Gy, with polymorphic band number 226 (83.7%). The total number of bands resulted from plant grew on 150 mM and 200 mM NaCl were 260 bands with polymorphic bands 185 (85.6%). However, the total number of bands produced from combined effects between gamma rays and salinity (20 Gy X 50 mM NaCl, 20 Gy X 100 mM NaCl and 25 Gy X 50 mM NaCl) were 270, with polymorphic band number 231 (85.5%). High similarity between treatments was revealed. Treatments relationships were estimated through cluster analysis (UPGMA) based on RAPD data. [ABSTRACT FROM AUTHOR]

In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S -, S -, and S -haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed. [ABSTRACT FROM AUTHOR]

Background and Aims Non-S-ribonucleases (non-S-RNases) are class III T2 RNases constitutively expressed in styles of species with S-RNase-based self-incompatibility. So far, no function has been attributed to these RNases. The aim of this work is to examine if NnSR1, a non-S-RNase from Nicotiana alata, is induced under conditions of phosphate (Pi) deprivation. The hypothesis is that under Pi-limited conditions, non-S-RNase functions may resemble the role of S-like RNases. To date, the only RNases reported to be induced by Pi deficiency are class I and class II S-like RNases, which are phylogenetically different from the class III clade of RNases. Methods Gene and protein expression of NnSR1 were assayed in plants grown hydroponically with and without Pi, by combining RT-PCR, immunoblot and enzymatic activity approaches. Key Results NnSR1 transcripts were detected in roots 7 d after Pi deprivation and remained stable for several days. Transcript expression was correlated based on Pi availability in the culture medium. Antiserum against a peptide based on a hypervariable domain of NnSR1 recognized NnSR1 in roots and stems but not leaves exposed to Pi shortage. NnSR1 was not detected in culture medium and was pelleted with the microsomal fraction, suggesting that it was membrane-associated or included in large compartments. The anti-NnSR1 inhibited selectively the enzymatic activity of a 31-kDa RNase indicating that NnSR1 was induced in an enzymatically active form. Conclusions The induction of NnSR1 indicates that there is a general recruitment of all classes of T2 RNases in response to Pi shortage. NnSR1 appears to have regained ancestral functions of class III RNases related to strategies to cope with Pi limitation and also possibly with other environmental challenges. This constitutes the first report for a specific function of class III RNases other than S-RNases. [ABSTRACT FROM PUBLISHER]

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes. [ABSTRACT FROM AUTHOR]

In tobacco (Nicotiana tabacum), nicotine and related pyridine alkaloids are produced in the root, and then transported to the aerial parts where these toxic chemicals function as part of chemical defense against insect herbivory. Although a few tobacco transporters have been recently reported to take up nicotine into the vacuole from the cytoplasm or into the cytoplasm from the apoplast, it is not known how the long-range translocation of tobacco alkaloids between organs is controlled. Nicotiana langsdorffii and N. alata are closely related species of diploid Nicotiana section Alatae, but the latter does not accumulate tobacco alkaloids in the leaf. We show here that N. alata does synthesize alkaloids in the root, but lacks the capacity to mobilize the root-borne alkaloids to the aerial parts. Interspecific grafting experiments between N. alata and N. langsdorffii indicate that roots of N. alata are unable to translocate alkaloids to their shoot system. Interestingly, genetic studies involving interspecific hybrids between N. alata and N. langsdorffii and their self-crossed or back-crossed progeny showed that the non-translocation phenotype is dominant over the translocation phenotype. These results indicate that a mechanism to retain tobacco alkaloids within the root organ has evolved in N. alata, which may represent an interesting strategy to control the distribution of secondary products within a whole plant. [ABSTRACT FROM PUBLISHER]

Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassette’ comprising 1,8-cineole, limonene, myrcene, α-pinene, β-pinene, sabinene, and α-terpineol. We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes, which produced the characteristic monoterpenes of this ‘cineole cassette’ with α-terpineol being most abundant in the volatile spectra. The amino acid sequences of both terpineol synthases were 99% identical. The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum. The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N. alata and N. langsdorfii were less efficient compared to the ‘cineole cassette’ monoterpene synthases of Arabidopsis thaliana, N. suaveolens, Salvia fruticosa, Salvia officinalis, and Citrus unshiu. The terpineol synthases of N. alata and N. langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals. The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N. alata was highest at the transition from day to night (diurnal rhythm). [ABSTRACT FROM PUBLISHER]

Self-incompatibility in the Solanaceae is mediated by S- RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S- RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S- RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S 5, S 27, S 70, S 75, S 107, and S 210, showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S- RNase expression. In contrast, the S 63 ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S 27, S 70, S 75, S 107, and S 210 alleles were fully functional novel S- RNases, while S 5 and S 63 resulted to be non- S- RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S- RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S- RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae. [ABSTRACT FROM AUTHOR]

The heritability and genetic basis of nectar traits have been rarely studied in the field, where plants are exposed to environmental factors that could mask underlying genetic effects. Heritabilities and variance components were estimated for nectar and morphological traits of Nicotiana alata, using a partial diallel design. The main experiment was conducted in a Missouri experimental garden using a randomized block design with three plant density treatments, whereas a smaller experiment was conducted near native Brazil habitat to compare the environmental variance in traits between Missouri and Brazil. Significant heritability was detected for nectar volume and energy content, and for corolla tube length. Phenotypic correlations were significant between all traits investigated, whereas significant genetic correlations were only found between nectar volume and energy and between corolla limb width and mouth diameter. There were no significant family-by-density interactions detected in the Missouri field environment. All traits differed significantly between Missouri and Brazil environments, but significant genetic by environment (G × E) interactions between Missouri and Brazil were detected for only one trait. This study shows that nectar traits can be heritable despite considerable environmental variation. [ABSTRACT FROM AUTHOR]

The NaGSL1 gene has been proposed to encode the callose synthase (CalS) enzyme from Nicotiana alata pollen tubes based on its similarity to fungal 1,3-β-glucan synthases and its high expression in pollen and pollen tubes. We have used a biochemical approach to link the NaGSL1 protein with CalS enzymic activity. The CalS enzyme from N. alata pollen tubes was enriched over 100-fold using membrane fractionation and product entrapment. A 220 kDa polypeptide, the correct molecular weight to be NaGSL1, was specifically detected by anti-GSL antibodies, was specifically enriched with CalS activity, and was the most abundant polypeptide in the CalS-enriched fraction. This polypeptide was positively identified as NaGSL1 using both MALDI-TOF MS and LC-ESI-MS/MS analysis of tryptic peptides. Other low-abundance polypeptides in the CalS-enriched fractions were identified by MALDI-TOF MS as deriving from a 103 kDa plasma membrane H+-ATPase and a 60 kDa β-subunit of mitochondrial ATPase, both of which were deduced to be contaminants in the product-entrapped material. These analyses thus suggest that NaGSL1 is required for CalS activity, although other smaller (<30 kDa) or low-abundance proteins could also be involved. [ABSTRACT FROM AUTHOR]

The pollen-specific promoter of the LAT52 gene is known to direct expression of marker proteins during the last stages of pollen maturation and in very early pollen tube growth.We have examined the expression of LAT52-GUS during later stages of pollen tube growth in style and ovary of the relatively long-styled species Nicotiana alata. GUS activity was detected histochemically and found to be present in germinating pollen grains of N. alata and in tubes growing through the upper part of the style. No GUS activity was detected in 99% of the pollen tubes growing through the lower part of the style, but activity was present in tubes within the ovary. This finding indicates that the LAT52 promoter is regulated in growing pollen tubes, and is most active during the earliest and latest stages of pollen tube growth. GUS activity was also detected in some ovules, where it presumably marked the release of pollen tube cytoplasm into the ovule. The distribution of ovules with GUS activity within the ovary is not consistent with high-precision pollen tube guidance to the ovule. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa PIs. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent. [ABSTRACT FROM AUTHOR]

The guidance of pollen tubes into the stigma, through the style and within the ovary of solanaceous plants is examined. New data based on time-lapse video microscopy of pollen on Nicotiana alata stigmas and in culture, as well as style squashes and pollinations with pollen bearing the marker gene Gus, are presented. The initial guidance of pollen tubes as they emerge from grains on the stigma is probably by a gradient in the concentration of water in the immediate environment of the pollen grain. At later stages of growth on the stigma, the probability that tube tips will encounter an opening is high because of the tendency of tubes to maintain contact with the surface. Styles support pollen tube growth either towards or away from the ovary, suggesting that they have no constitutive directionality. Guidance within solid transmitting tracts is probably by default, i.e. the architecture of the tissue means that pollen tubes can only grow in the intercellular spaces between the parallel files of cells. Pollen tubes within the ovary of N. alata do not appear to be precisely guided to the nearest ovule, and further, quantitative studies of growth in this region are needed. [ABSTRACT FROM AUTHOR]

Nicotiana alata is an ornamental horticultural plant with a variety of flower colors and a long flowering period. The genes in four different colored N. alata (white, purple, red, and lemon green) were analyzed to explain the differences in flower color using transcriptomes. A total of 32 differential expression genes in the chlorophyll biosynthesis pathway and 41 in the anthocyanin biosynthesis pathway were identified. The enrichment analysis showed that the chlorophyll biosynthesis pathway and anthocyanin biosynthesis pathway play critical roles in the color differences of N. alata. The HEMA of the chlorophyll biosynthesis pathway was up-regulated in lemon green flowers. Compared with white flowers, in the red and purple flowers, F3H, F3′5′H and DFR were significantly up-regulated, while FLS was significantly down-regulated. Seventeen differential expression genes homologous to transcription factor coding genes were obtained, and the homologues of HY5, MYB12, AN1 and AN4 were also involved in flower color differences. The discovery of these candidate genes related to flower color differences is significant for further research on the flower colors formation mechanism and color improvements of N. alata. [ABSTRACT FROM AUTHOR]

Many flowering plants have developed a self-incompatibility mechanism, which is controlled by a single polyallelic locus (the S-locus), to prevent inbreeding. The products of the S-locus in file styles of solanaceous plants are an allelic series of glycoproteins with RNase activity [McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F. & Clarke, A. E. (1989) Nature 342, 955– 957]. These S-RNases show some amino-acid-sequence similarity with two fungal RNases (T2 and Rh), including the presence of two active-site His residues, which suggests a common three-dimensional structure. Disulphide bonding is important in the maintenance of the three-dimensional structure of the fungal RNases [Kurihara, H., Mitsui, Y., Ohgi, K., Irie, M., Mizuno, H. & Nakamura, T. (1992) FEBS Lett. 306, 189–1921 and the S-RNases [Tsai, D. S., Lee, H.-S., Post, L. C., Kreiling, K. M. & Kao, T.-H. (1992) Sex. Plant Reprod. 5, 256–263]. We have used the S2-allele RNase of Nicotiana alata, which has nine Cys residues, to establish the pattern of disulphide bonding. The disulphide bonds Cysl6–Cys21, Cys45–Cys94, Cys153–Cys182 and Cys165–Cys176 are consistent with the S2-RNase having a similar three-dimensional structure to RNase Rh. A free Cys residue (Cys95) adjacent to Cys45–Cys94 promotes a rapid specific disulphide migration when the protein is exposed to denaturing conditions. [ABSTRACT FROM AUTHOR]

Some members of the Solanaceae have a self‐incompatibility mechanism preventing self‐fertilization. Stylar ribonucleases (S‐RNases) are responsible for growth inhibition of self‐pollen tubes. A prevalent model postulates that the S‐RNases act as intracellular cytotoxins that degrade ribosomal RNA, and possibly also messenger RNA, in the incompatible pollen tubes. Since ribosomes and polysomes are easily noticed with the electron microscope, it should be possible to confirm disintegration of these structures. However, our inspection by electron microscopy revealed the presence of ribosomes and polysomes in pollen tubes formed after self‐pollination of the self‐sterile species Brugmansia (Datura) suaveolens and Nicotiana alata. There was no decrease over time in the number of bound ribosomes per unit of rough endoplasmic reticulum (RER) membrane. The results indicate that the inhibition of tube growth is not due to a general degradation of ribosomal and messenger RNA. Therefore, the substrate for S‐RNases presumably is very specific. [ABSTRACT FROM AUTHOR]