Differential Gene Expression with an Emphasis on Floral Organ Size Differences in Natural and Synthetic Polyploids of Nicotiana tabacum (Solanaceae).

Floral organ size, especially the size of the corolla, plays an important role in plant reproduction by facilitating pollination efficiency. Previous studies have outlined a hypothesized organ size pathway. However, the expression and function of many of the genes in the pathway have only been investigated in model diploid species; therefore, it is unknown how these genes interact in polyploid species. Although correlations between ploidy and cell size have been shown in many systems, it is unclear whether there is a difference in cell size between naturally occurring and synthetic polyploids. To address these questions comparing floral organ size and cell size across ploidy, we use natural and synthetic polyploids of Nicotiana tabacum (Solanaceae) as well as their known diploid progenitors. We employ a comparative transcriptomics approach to perform analyses of differential gene expression, focusing on candidate genes that may be involved in floral organ size, both across developmental stages and across accessions. We see differential expression of several known floral organ candidate genes including ARF2, BIG BROTHER, and GASA/GAST1. Results from linear models show that ploidy, cell width, and cell number positively influence corolla tube circumference; however, the effect of cell width varies by ploidy, and diploids have a significantly steeper slope than both natural and synthetic polyploids. These results demonstrate that polyploids have wider cells and that polyploidy significantly increases corolla tube circumference.

Bioinformatics study of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) genes in Solanaceae.

Isoprenoids, the largest and most diverse class of secondary metabolites in plants, play an important role in plant growth and development. Isoprenoids can be synthesized by two distinct pathways: the methylerythritol-4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first step and a key regulatory enzyme of the MEP pathway in plants. The DXS gene has been reported to play a key role in seedling development, flowering, and fruit quality in plants of the Solanaceae, such as tomato, potato and tobacco. However, to improve our understanding and utilization of DXS genes, a thorough bioinformatics study is needed. In this study, 48 DXS genes were aligned and analyzed by computational tools to predict their protein properties, including molecular mass, theoretical isoelectric point (pI), signal peptides, transmembrane and conserved domains, and expression patterns. Sequence comparison analysis revealed strong conservation among the 48 DXS genes. Phylogenetic analysis indicated that all DXS genes were derived from one ancestor and could be classified into three groups with different expression patterns. Moreover, the functional divergence of DXS was restricted after gene duplication. The results suggested that the function and evolution of the DXS gene family were highly conserved and that the DXS genes of Group I may play a more important role than those of other groups.

Effects of living hyperaccumulator plants and their straws on the growth and cadmium accumulation of Cyphomandra betacea seedlings.

To determine whether the living hyperaccumulator plants and their straws have the same effects on the growth and heavy metal accumulation of common plants, two pot experiments (intercropping experiment and straw mulch experiment) were conducted to study the effects of living hyperaccumulator plants (Solanum photeinocarpum, Tagetes erecta, Galinsoga parviflora and Bidens pilosa) and their straws on the growth and cadmium (Cd) accumulation of common plant Cyphomandra betacea seedlings. Intercropping with T. erecta or B. pilosa promoted the growth of C. betacea seedlings compared with the monoculture, while intercropping with S. photeinocarpum or G. parviflora inhibited that. Intercropping with S. photeinocarpum decreased the Cd contents in the roots and shoots of C. betacea seedlings compared with the monoculture, but intercropping with the other plants did not. In the straw mulch experiment, the straw of S. photeinocarpum or T. erecta promoted the growth of C. betacea seedlings compared with the control, while the straw of G. parviflora or B. pilosa did not. The straw of S. photeinocarpum or T. erecta decreased the Cd contents in the shoots of C. betacea seedlings, and the straw of G. parviflora or B. pilosa increased the shoot Cd contents. Thus, intercropping with S. photeinocarpum and applying S. photeinocarpum or T. erecta straw can reduce the Cd uptake of C. betacea.

Plant reference genes for development and stress response studies.

Many reference genes are used by different laboratories for gene expression analyses to indicate the relative amount of input RNA/DNA in the experiment. These reference genes are supposed to show least variation among the treatments and with the control sets in a given experiment. However, expression of reference genes varies significantly from one set of experiment to the other. Thus, selection of reference genes depends on the experimental conditions. Sometimes the average expression of two or three reference genes is taken as standard. This review consolidated the details of about 120 genes attempted for normalization during comparative expression analysis in 16 different plants. Plant species included in this review are Arabidopsis thaliana, cotton (Gossypium hirsutum), tobacco (Nicotiana benthamiana and N. tabacum), soybean (Glycine max), rice (Oryza sativa), blueberry (Vaccinium corymbosum), tomato (Solanum lycopersicum), wheat (Triticum aestivum), potato (Solanum tuberosum), sugar cane (Saccharum sp.), carrot (Daucus carota), coffee (Coffea arabica), cucumber (Cucumis sativus), kiwi (Actinidia deliciosa) and grape (Vitis vinifera). The list includes model and cultivated crop plants from both monocot and dicot classes. We have categorized plant-wise the reference genes that have been used for expression analyses in any or all of the four different conditions such as biotic stress, abiotic stress, developmental stages and various organs and tissues, reported till date. This review serves as a guide during the reference gene hunt for gene expression analysis studies.

Identification and expression profiling of DNA methyltransferases during development and stress conditions in Solanaceae.

DNA methyltransferase (DMTase) enzymes contribute to plant development and stress responses by de novo establishment and subsequent maintenance of DNA methylation during replication. However, the molecular mechanism underlying this activity remains obscure, especially in crop species. Using DMTase homolog complement in six Solanaceae species, we demonstrated here that their number remained conserved in Solanum lineage, whereas it was expanded in both pepper and Nicotiana benthamiana. Non-synonymous vs synonymous (Ka/Ks) substitution ratio revealed that most of the Solanaceous DMTase homologs undergo purifying selection. The genomic sequences of tomato DMT homologs in its wild relative, Solanum pennellii, remained highly conserved in their exons and methyltransferase domains. Structure analysis further revealed highly similar folding of DMTase homologs and conservation in the residues participating in protein-protein interaction in Solanum lineage, whereas a considerable diversification was observed of pepper homologs. Transcript profiling of DMTases highlighted both similar and distinct expression patterns of tomato homologs in other species during fruit development and stress responses. Overall, our analysis provides a strong basis for in-depth exploration of both conserved as well as distinct functions of tomato DMTase homologs in other economically important Solanaceae species.

Androgenesis in Solanaceae.

The Solanaceae is one of the most important families for global agriculture. Among the different solanaceous species, tobacco (Nicotiana tabacum), potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum) are five crops of outstanding importance worldwide. In these crops, maximum yields are produced by hybrid plants created by crossing pure (homozygous) lines with the desired traits. Pure lines may be produced by conventional breeding methods, which is time consuming and costly. Alternatively, it is possible to accelerate the production of pure lines by creating doubled haploid (DH) plants derived from (haploid) male gametophytes or their precursors (androgenesis). In this way, the different steps for the production of pure lines can be reduced to only one generation, which implies important time and cost savings. This and other advantages make androgenic DHs the choice in a number of important crops where any of the different experimental in vitro techniques (anther culture or isolated microspore culture) is well set up. The Solanaceae family is an excellent example of heterogeneity in terms of response to these techniques, including highly responding species such as tobacco, considered a model system, and tomato, one of the most recalcitrant species, where no reliable and reproducible methods are yet available. Interestingly, the first evidence of androgenesis, particularly through in vitro anther culture, was demonstrated in a solanaceous species, Datura innoxia. In this chapter, we report the state of the art of the research about androgenic DHs in Solanaceae, paying special attention to datura, tobacco, potato, tomato, eggplant, and pepper.

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species.

UNLABELLED: Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. KEY MESSAGE: SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.

Comparative anatomical and developmental analysis of dry and fleshy fruits of Solanaceae.

PREMISE OF THE STUDY: An anatomical examination of dry and fleshy fruits within the Solanaceae was carried out to identify comparable stages throughout development as well as features exclusive to each type of fruit. We studied fruit development of Nicotiana and Petunia, which have the plesiomorphic capsular fruit; Solanum and Iochroma, characterized by a derived fleshy berry; Cestrum, an independent origin of a fleshy fruit; and Datura, a reversion to a dry fruit. • METHODS: Pre- and postanthesis carpels and fruits of all species were collected, sectioned, stained, and examined using light microscopy. • KEY RESULTS: Comparable stages of carpel and fruit development were identified in all species. Furthermore, anatomical and developmental features were identified that characterize capsules in Solanaceae, including lack of increase in the number of pericarp cell layers, formation of a sclerified endocarp, and elongation of the epidermal cells of the placenta. Pericarps of fleshy fruits of the Solanoideae are characterized by abundant collenchyma, an increase in the number of cell layers, and a parenchymatous endocarp often expanding into the locules. Anatomical data show that early developmental stages of the fruit of Cestrum, a berry, are similar to the capsular fruits of Petunia and Nicotiana; similarly, Datura, one of the few capsular members of the Solanoideae, shares several anatomical features with closely related berried taxa. • CONCLUSIONS: Ontogenetically, all fleshy or all dry fruits do not necessarily share a common developmental ground plan. Independent evolution of fleshiness, sclerification, dryness, and dehiscence are discussed in a phylogenetic context.

Correlação entre floração, frutificação e variáveis ambientais em Solanum lycocarpum. A. St. Hil, Solanaceae / Correlation between flowering, fructification and environmental variables in Solanum lycocarpum A. St.-Hil, Solanaceae

A lobeira (S. lycocarpum) é uma espécie típica e abundante do Cerrado, que ocupa principalmente ambientes antropizados. Possui características interessantes do ponto de vista da biologia reprodutiva, que provavelmente estão favorecendo a ampla ocupação pela espécie. Tendo em vista que a planta floresce e frutifica durante todo o ano, o presente estudo teve por objetivo verificar se existe correlação entre floração, frutificação e variáveis ambientais (temperatura, umidade e precipitação), como suporte para estudos futuros referentes à biologia reprodutiva e ecologia de espécies de plantas do Cerrado. Foi escolhida uma população de lobeira situada no município de Morrinhos, Sul do estado de Goiás, composta de 34 plantas em fase reprodutiva. Todas as plantas foram georreferenciados com um aparelho GPS. Foram feitas observações mensais durante 13 meses (junho de 2005 a julho de 2006) e quantificados flores abertas e frutos produzidos nos intervalos entre observações. Pode-se inferir alta conversão de flores hermafroditas em frutos. Por meio da correlação de Spearman, pode-se constatar correlação positiva entre floração e precipitação e umidade, o mesmo, aparentemente, não ocorre para frutificação.

Solanum lycocarpum (lobeira) is a typical and abundant species of brazilian Cerrado, which occupies mainly surrounding disturbed areas. It has interesting characteristics from the point of view of reproductive biology, that probably are favoring the large occupation of habitats by the species. Based on the fact that the species produces flowers and fruit during all the year, the present study had the purpose to verify the association between flower and fruit production with environmental variables (temperature, relative humidity and precipitation), aiming to support future studies referring to reproductive biology and ecology of plant species from Cerrado biome. A population of S. lycocarpum composed of 34 plants in reproductive phase, situated in Morrinhos, south of the State of Goiás, Brazil, , was evaluated. All the plants were geographically referenced with a GPS receptor. Observations were made monthly during 13 months (June, 2005 to July, 2006) quantifying open flowers and fruits produced in the intervals between the observations. It was possible suggest high conversion of flowers in fruits. The Spearman rank correlation showed positive correlation of flower number with precipitation and relative humidity. Fruit number was not correlated with the environmental variables.

Solasodine accumulation in regenerated plants of Solanum torvum Sw / Acúmulo de solasodina em plantas micropropagadas de Solanum torvum Sw

A nodal segment culture was developed in order to assess Solanum torvum Sw. regeneration and solasodine levels. The influence of auxins (indoleacetic acid, 1-Naphthaleneacetic acid) and benzyl adenine on S. torvum growth in micropropagation was investigated. A nodal segment culture was initiated with seeds germinated in MS basal medium added of GA3 and grown in different concentrations of IAA, IAA + BAP and NAA + BAP. Sixty-day-old plants from the in vitro culture were collected, frozen and lyophilized; then, the methyl orange method was used to quantify solasodine for the spectrophotometric assay. The best results regarding plant regeneration and solasodine accumulation were obtained by using the MS basal medium without addition of plant growth regulators; however, there was great production of calluses presenting friable bases. Based on these results, cell cultures can be initiated from such calluses with application of other auxins and cytokinins to enhance solasodine production, besides different elicitors, light intensities and sucrose concentrations.

A regeneração de Solanum torvum e a avaliação do conteúdo de solasodina foram os objetivos de cultura de segmentos nodais. A influência de auxinas (ácido 3-indolacético, ácido naftalenoacético) e de 6-benzilaminopurina no crescimento de S. torvum na micropropagação foi investigado. Cultura de segmentos nodais foi iniciada por sementes germinadas em meio básico MS acrescido de GA3 e cultivadas em diferentes concentrações de AIA, AIA + BAP e ANA + BAP. Plantas da cultura in vitro com 60 dias foram coletadas, congeladas e liofilizadas e o método de alaranjado de metila foi utilizado para quantificação de solasodina para o ensaio espectrofotométrico. Os melhores resultados para regeneração vegetal e acúmulo de solasodina foram alcançados no meio MS sem adição de reguladores de crescimento havendo, porém grande produção de calos de base friáveis. Esses resultados mostram que a partir desses calos, cultura de células pode ser iniciada com aplicação de outras auxinas e citocininas para o aumento da produção de solasodina além de diferentes eliciadores, intensidades luminosas e concentrações de sacarose.

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

How polyamine synthesis inhibitors and cinnamic acid affect tropane alkaloid production.

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhydrazone (MGBG) and the phenylalanine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250- 1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.

Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species.

Here, orthologous genes of six phosphate transporter (PiT) genes, which are members of the Pht1 and Pht2 families in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns to those in tomato and potato: P-starvation enhancement in both leaves and roots for Pht1;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant. In addition, the mycorrhizal symbiosis down-regulated the expression of Pht2;1 genes greatly in the leaves of pepper and tobacco. The discrepancies between the evolutionary distances of the PiT genes and their expression patterns among the five species suggest greater complexity in function of PiT in plants than previously expected.

Trichomes: different regulatory networks lead to convergent structures.

Sometimes, proteins, biological structures or even organisms have similar functions and appearances but have evolved through widely divergent pathways. There is experimental evidence to suggest that different developmental pathways have converged to produce similar outgrowths of the aerial plant epidermis, referred to as trichomes. The emerging picture suggests that trichomes in Arabidopsis thaliana and, perhaps, in cotton develop through a transcriptional regulatory network that differs from those regulating trichome formation in Antirrhinum and Solanaceous species. Several lines of evidence suggest that the duplication of a gene controlling anthocyanin production and subsequent divergence might be the major force driving trichome formation in Arabidopsis, whereas the multicellular trichomes of Antirrhinum and Solanaceous species appear to have a different regulatory origin.

Induced defense in Nicotiana attenuata (Solanaceae) fruit and flowers.

Plants protect themselves against herbivory using a continuum of strategies, ranging from constitutive defenses to intermittent induced responses. Induced defenses may not provide immediate and maximum protection, but could be advantageous when continuous defense is either energetically or ecologically costly. As such, induced defenses in flowers could help defend relatively valuable tissue while keeping reproductive structures accessible and attractive to pollinators. Thus far, no one has demonstrated the efficacy of induced defenses against floral herbivores (florivores) in the field. Here we show that mechanical leaf damage in wild tobacco, Nicotiana attenuata (Solanaceae), reduced both flower and fruit herbivory in the field and that exogenous application of methyl jasmonate, a potent elicitor of induced responses, reduced both leaf and floral damage in natural populations. This result is consistent with a survey of damage in the field, which showed a negative relationship between leaf damage and flower and fruit damage. Although optimal defense theory predicts that induced defenses should be rare in reproductive tissues, owing to their high fitness value, our results suggest otherwise. Induced defenses in leaves and reproductive tissues may allow plants to respond effectively to the concomitant pressures of defending against herbivory and attracting pollinators.

Tobacco to tomatoes: a phylogenetic perspective on fruit diversity in the Solanaceae.

The Solanaceae contains many species of agricultural importance. Several of these are cultivated for their fruits, such as the tomato, the pepper and the aubergine. The family is very diverse in fruit type with capsules, drupes, pyrenes, berries, and several sorts of dehiscent non-capsular fruits occurring in the 90+ genera. In this paper, recent work on fruit type evolution in angiosperms is reviewed in relation to dispersal agents and habitat ecology. Defining fruit types in the Solanaceae in a simple five state system, then mapping them onto a previously published molecular phylogeny based on chloroplast DNA allows discussion of the evolution of these fruit types in a phylogenetic framework. Capsules are plesiomorphic in the family, and although berries are a synapomorphy (shared derived character) for a large clade including the genus Solanum (tomatoes and aubergines), they have arisen several times in the family as a whole. Problems with homology of drupes and pyrenes are discussed, and areas for future investigation of fruit structure homology identified. The distribution of fruit types in the large and diverse genus Solanum is also discussed in the light of monophyletic groups identified using chloroplast gene sequences. This variety is related to recent advances in the understanding of the molecular biology of fruit development. Finally, several key areas of future comparative, phylogenetic investigation into fruit type evolution in the family are highlighted.

Ethylene response to pollen tube growth in Nicotiana tabacum flowers.

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression.

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-0-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.

Conservation in divergent solanaceous species of the unique gene structure and enzyme activity of a gametophytically-expressed flavonol 3-O-galactosyltransferase.

Flavonol 3-O-galactosyltransferase (F3GalTase) is a pollen-specific enzyme which glycosylates the flavonols required for germination in petunia. The highly restricted tissue-specific expression and substrate usage make F3GalTase unique among all other flavonoid glycosyltransferases (GTs) described to date, including the well characterized Bronze 1 (Bz1) gene of maize. RFLP mapping, DNA gel blot, and sequence analyses showed that the single copy F3galtase gene has a different genomic organization than Bz1. Within the promoter of F3galtase are potential regulatory motifs which may confer pollen-specific gene expression and activation by Myb and bHLH transcription factors. However, we provide evidence that F3galtase is not regulated by An4, which encodes a Myb factor known to regulate anthocyanin accumulation in petunia anthers. An unexpected feature of the F3galtase promoter was the presence of large blocks of chloroplast and mitochondrial DNA. Gel blot analyses of genomic DNA from the progenitors of Petunia hybrida, as well as from Nicotiana tabacum, indicated that migration of organellar DNA into the F3galtase gene was an ancient event that occurred prior to speciation of the Solanaceae. Together with enzyme assays and HPLC analyses of pollen extracts from tobacco, tomato, and potato, these results confirmed that the unique F3galtase gene structure, enzyme activity, and pollen-specific flavonol glucosylgalactosides are conserved throughout the Solanaceae.

Root formation in ethylene-insensitive plants.

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.