Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Leaf-Mediated Light Responses in Petunia Flowers

In the present work we studied the role of light in the regulation of flavonoid gene expression and anthocyanin synthesis in petunia (Petunia hybrida) corollas. We found that light is required for chalcone synthase gene (chs) expression, anthocyanin synthesis, and growth of detached and attached petunia corollas. Although direct illumination induced chs expression, pigmentation, and elongation of the detached corollas, irradiation of green leaves or sepals played the main role in the attached corollas. The duration, intensity, and spectrum of the light reaction suggest that phytochrome-mediated high-irradiance reactions are involved in the regulation of corolla development. Using the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, we showed that photosynthesis does not significantly contribute to the leaf-mediated light responses. When sepals were removed or covered. [14C]sucrose up-take by the corolla of detached intact flowers was inhibited. The results of this study suggest that light is perceived by leaves and sepals and enhances corolla sink activity, elongation, pigmentation, and chs expression. The role of leaves and sepals in the light regulation of petunia corolla development is discussed.     

Temporal, spatial and hormonal regulation of the S-adenosylmethionine synthetase gene in petunia

Gibberellic acid (GA₃) promotes corolla elongation and pigmentation in petunia flowers. We have previously shown that G.A₃ induces pigmentation by activating specific genes of the anthocyanin biosynthetic pathway. The aim of the present work was to examine whether GA₃ induces also the expression of genes from other metabolic pathways in petunia corollas that may be associated with growth. Recently we reported the cloning of the petunia sam gene coding for S -adenosylmethionine synthetase (SAM-S). In the present work we show that sam expression is induced by GA₃ in both corollas and stems. The expression of the gene was correlated with corolla elongation. GA₃ and the cylokinin, N -6-benzyladenine (BA) promoted corolla growth and sam expression, whereas abscisic acid (ABA) inhibited corolla elongation and repressed sam mRNA accumulation. An analysis of sam expression in stems indicated a high level in young, elongating internodes and a very low level in the mature, non-elongating stem zone. The results of the present study show that the effect of GA₃ on gene expression in the corolla of petunia, is not restricted to the anthocyanin biosynthetic pathway, they also suggest a possible role for sam in GA₃-induced corolla and stem elongation.     

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.     

The petunia homologue of tomato gast1: transcript accumulation coincides with gibberellin-induced corolla cell elongation

Gibberellins (GAs) regulate petunia corolla pigmentation and elongation. To study this hormone's effect at the molecular level, we used the tomato gast1 gene as a probe to isolate a gibberellin-induced gene (gip) from petunia corollas. The deduced sequence of gip exhibited 82% identity with GAST1 protein and contained a short, highly hydrophobic N-terminal region. High levels of gip expression were detected in elongating corollas and young stem internodes. When detached corollas were grown in vitro in sucrose medium, gip expression was strongly induced by gibberellic acid (GA₃). GA₃-induced gip expression in corollas was inhibited by abscisic acid (ABA). The expression of the gene was also induced by GA₃ in detached young stem segments. Sucrose was not essential for GA-induced gip expression in corollas but enhanced its effect. In stems, on the other hand, sucrose inhibited the effect of the hormone. The results of the present work support the possible role of gip in GA-induced corolla and stem elongation.     

Photosynthetic carbon fixation in the corollas of Petunia hybrida

Corollas of Petunia hybrida (cv. Hit Parade Rosa) flowers fixed ¹⁴CO₂ under both light and dark conditions. Rates of light fixation were much higher in mature pink corollas than in young, green corollas [57 and 9 nmol (ngchl)¹ min^-1], paralleling the development of chloroplasts in these tissues. Stomatal conductance in corollas was only 12% of that in green leaves, mainly due to the presence of few, and non-functioning stomata in the corolla. The activity and concentration of ribulose bisphosphate carboxylase (EC 4.1.1.39) in corolla extracts were only about 30% (per unit Chi) of those in extracts from green leaves. These results, together with previous results, might indicate a coordinated reduction in activity of systems participating in photosynthesis in corollas. The fixation products following a 6 s pulse with ¹⁴CO₂, were typical of C, plants in both corollas and green leaves, but a higher level of β-carboxylation products was found in the corollas. The activity of phosphoenol-pyruvate carboxylase (EC 4.1.1.31) (per unit protein) was similar in both tissues. Although the total carbon fixed by the corolla constituted only a small part of the metabolites required for flower development, certain photosynthetic metabolites might have a regulatory role in flower development.     

Photosynthetic activities in the petunia corolla

Pink Petuniahybrida (cv Hit Parade Rosa) corollas were found to contain photosynthetically active chloroplasts. The corolla chloroplasts were similar to those of green leaves in size and structure. The chlorophyll (Chl) content of Petunia corollas increased during early stages of flower development, reaching a maximum just before anthesis. Chloroplasts isolated from corollas at this stage, carried out photosystem I-dependent electron transport at rates which were two-thirds of those measured in chloroplasts from green leaves, but full chain electron transport at only one-quarter of the rate carried out by chloroplasts from green leaves. Both the light saturated rate and the quantum yield for electron transport were lower in corolla chloroplasts, which also required lower intensities for light saturation. Reduced efficiency of photosystem II photoreactions in the corolla was also indicated by the ratio between variable and constant components of Chl fluorescence, which was lower in corollas compared to green leaves. The induction time of Chl fluorescence was at least three times shorter in corollas compared to green leaves, indicating a smaller number of functional photosystem II centers (per Chl) in the corolla. It is suggested that corolla chloroplasts of Petunia might have a role in flower developmental processes.     

Disproportionate carbon and water maintenance costs of large corollas in hot Mediterranean ecosystems

Larger corollas increase the reproductive success of entomophilous plants, but are also associated with increased carbon and water costs, especially under hot and dry conditions. Minimizing floral carbon and water loss by reducing corolla size should be potentially advantageous for plants living in these environments. We quantify maintenance costs of corollas (water and carbon) in large-flowered rockroses (Cistus spp.) in a Mediterranean ecosystem. We performed field studies of two coflowering sympatric Cistus of contrasting corolla size to analyze water costs. Additionally, we used the larger-flowered species (C. ladanifer) to analyze the effects of intraspecific variation in corolla size on floral net carbon exchange and transpiration rates. We also assessed the mean daily percentage of plant water and carbon consumed by corollas by comparing with that of leaves at the time of flowering in C. ladanifer. Temperature and corolla area increased water maintenance costs, following an allometric relationship where transpiration rate per unit of area increased with corolla area. Larger flowers tended to heat less under strong irradiance than smaller ones in both species, especially in C. ladanifer, demonstrating a stronger transpirational cooling effect on larger flowers. In terms of carbon, temperature significantly affected net carbon exchange rates, which were not affected by corolla size. Daily water and carbon expenses of corolla were ca. 50% of those of leaves on an organ surface area basis. Our results suggest that water and carbon maintenance costs of large flowers in the Mediterranean impose significant constraints to corolla size, ecophysiologically favoring smaller-flowered individuals in these ecosystems.     

Gibberellin regulates post-microsporogenesis processes in petunia anthers

Previous studies have suggested that gibberellins (GAs) are produced in petunia anthers and transported to the corolla to induce growth and pigmentation. In this work, we studied the role of GA in the regulation of anther development. When petunia plants were treated with the GA-biosynthesis inhibitor paclobutrazol, anther development was arrested. Microscopic analysis of these anthers revealed that paclobutrazol inhibits post-meiotic developmental processes. The treated anthers contained pollen grains but the connective tissue and tapetum cells were degenerated. A similar phenotype was obtained when the Arabidopsis GA-signal repressor, SPY, was over-expressed in transgenic petunia plants, i.e. anther development was arrested following microsporogenesis. The expression of the GA-induced gene, GIP , can be used in petunia as a molecular marker to study GA responses. GA₃ treatment of young anthers promoted, and paclobutrazol inhibited, GIP expression, suggesting that the hormone controls the natural activation of the gene in the anthers. Analyses of GIP expression during anther development revealed that the gene is induced only after microsporogenesis. This observation further suggests a role for GA in the regulation of post-meiotic processes during petunia anther development.     

Respiration, Carbon Balance and Translocation of Dry Matter in the Corolla of Rose Flowers

The effects of import of dry matter from the leaves on respiration,metabolism of carbohydrates and development of the corolla werestudied in intact flowering rose shoots and compared with cutflowering shoots bearing either seven leaves on 45 cm stemsor two leaves on 30 cm stems. Over 4 days, corollas of two-leaf shoots imported and respiredless carbon than either the intact or the seven-leaf shootsand they developed more slowly. Starch levels in the corollaat the end of this time depended on the stage at which it wascut; more advanced flowers (stage 3–4) tended to losestarch whereas less advanced ones (stage 1–2) tended toaccumulate it. Total carbohydrate in the corolla was dependenton leaf number. Loss of d. wt of leaves indicated that they provided dry matterto the corollas even in cut shoots, and this observation wassupported by the recovery of ¹⁴C-leaf assimilate from corollas.The depletion of dry matter from leaves and the slight delayin corolla development of the two-leaf cut shoots suggest thata dynamic source-sink (leaves—corolla) relationship existsin the senescing cut rose as in the intact rose. Rosa sp, corolla, respiration, carbon balance, translocation     

Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas

The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent.     

Nitrogen metabolism and flower symmetry of petunia corollas treated with glyphosate

A change of flower shape was observed in petunia corollas treated with 0.5 mM glyphosate. Glyphosate changed the flower symmetry from the actinomorphic type to the zygomorphic type. Corollas treated with glyphosate showed an increased free amino acid content. Free amino acid profiles in petunia corollas revealed that glyphosate had no significant effect on aromatic amino acid levels but increased the level of proline. Soluble protein content in glyphosate-treated corollas did not cause any significant changes. The contents of soluble phenolics, lignin, and IAA in the corollas were not significantly affected by the glyphosate treatment. In contrast, glyphosate reduced the nitrate content and the RNA content of petunia corollas by 45% and 63% of the control, respectively. However, the DNA content in glyphosate-treated corollas was similar to that of the control. Low concentrations of glyphosate did not show any phytotoxic effects on the whole plants and any remarkable changes on aromatic amino acid metabolism and protein synthesis. However, glyphosate reduced the RNA content of petunia corollas and changed the flower symmetry from the actinomorphic type to the zygomorphic type. The results of nonprotein nitrogen metabolism in glyphosate-treated petunia corollas suggested that glyphosate application at low concentration may influence the regulation of flower symmetry through the change of RNA biosynthesis.     

粪透明颤菌血红蛋白基因促进转基因矮牵牛在水培条件下生长并增强其抗涝能力

通过PCR程序克隆粪透明颤菌（Vitreoscilla steroraria Pringsheim)血红蛋白基因（vhb)的编码区。将其置于CaMV35S启动子的驱动下导入矮牵牛（Petunia hybriad Vilm),PCR和Southern杂交分析证明vhb基因已整合到受体基因组中,而RT-PCR检测证实了转基因矮牵牛中vhb基因转录水平的表达。观察了转基因株系在液体培养基中对淹水和缺氧的反应,发现vhb的表达明显促进了水培植物的生长。为进一步研究转基因植物在低氧条件的耐受能力。将上述的转基因株系在静置的液体培养基中进行完全的淹没培养,结果显示转基因植株具有较强的低氧耐受能力。培养两周后能从淹没状态长出液面,并由此而在液体中较正常地生长,而未转基因的对照不能长出淹没的培养基表面,在4-5周后因缺氧而窒息死亡。另将上述转基因株系在温室中盆栽并进行抗涝分析。在模拟的持续淹水胁迫中,转基因株系比对照表现出较强的忍受能力。这些结果预示vhb基因在抗涝作物培育和提高水培植物缺氧耐受能力的分子育种方面具有较良好的应用前景。