A gene controlling rate of anthocyanin synthesis and mutation frequency of the gene An1 in Petunia hybrida

Summary The difference in colour intensity between flowers of sporogenic revertants of the white flowering lines W17 and W28 is caused by an incompletely dominant gene Inl. This gene is not linked to the anthocyanin gene Anl. In the dominant state Inl causes a 50% decrease in colour intensity of selfcoloured red flowers.Chromatographic analysis of anthocyanins of plants homozygous recessive or dominant for Inl showed that the same anthocyanins are produced in both genotypes (cyanidin-3-glucoside and cyanidin-3-diglucoside). Anthocyanin synthesis starts at the same stage of development of the flower in both genotypes. When the bud reaches a length of approximately 45 mm, however, anthocyanin synthesis in the Inl Inl line slows down.No influence of the gene Inl on the concentration of dihydroquercetin-7-glucoside in buds and flowers could be observed, which indicates that the influence of Inl on flower colour development is restricted to the last part of the biosynthesis of anthocyanins, i.e. the conversion of dihydroflavonols into anthocyanins.In addition to Inl having a decreasing effect on flower colour intensity, evidence is produced that the gene Inl also influences the reversion frequency of unstable alleles of the gene Anl.     

Quantitative analysis for the cellulose I alpha crystalline phase in developing wood cell walls

FT-IR and X-ray analyses were employed to determine the relative ratio of cellulose Ialpha and Ibeta crystalline phases present in each developmental stage of coniferous tracheid cell wall formation. The IR spectra showed that initially the Ialpha phase occupies 50% of the crystalline regions in the primary cell wall cellulose and this value drops to 20% after ceasing of the cell enlarging growth for the formation of the secondary wall cellulose (the remaining regions are composed of the Ibeta phase). Although it is reasonable that the content for Ibeta, which is stress-reduced crystalline form, was higher in the secondary wall formation (Kataoka Y, and Kondo T. Macromolecules 1996;29:6356 6358) it is more interesting that during the crystallization of stress-induced Ialpha cellulose for the primary wall the stress-reduced Ibeta, is also possible to be crystallized in an alternative way. This means that throughout the period the Ialpha-causing stress may not be necessarily kept loaded. In light of our previously reported hypothesis (Kataoka Y. and Kondo T. Macromolecules 1998;31:760-764) for the formation of Ialpha phase due to cellular growing stresses in the primary wall cellulose, such an alternating on-off stress effect to account for the occurrence of both Ialpha and Ibeta phases might be related to a biological growth system in coniferous wood cells.     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

Over-expression of the Gerbera hybrida At-SOC1-like1 gene Gh-SOC1 leads to floral organ identity deterioration

Background and Aims

The family of MADS box genes is involved in a number of processes besides controlling floral development. In addition to supplying homeotic functions defined by the ABC model, they influence flowering time and transformation of vegetative meristem into inflorescence meristem, and have functions in roots and leaves. Three Gerbera hybrida At-SOC1-like genes (Gh-SOC1–Gh-SOC3) were identified among gerbera expressed sequence tags.

Methods

Evolutionary relationships between SOC1-like genes from gerbera and other plants were studied by phylogenetic analysis. The function of the gerbera gene Gh-SOC1 in gerbera floral development was studied using expression analysis, protein–protein interaction assays and reverse genetics. Transgenic gerbera lines over-expressing or downregulated for Gh-SOC1 were obtained using Agrobacterium transformation and investigated for their floral phenotype.

Key Results

Phylogenetic analysis revealed that the closest paralogues of At-SOC1 are Gh-SOC2 and Gh-SOC3. Gh-SOC1 is a more distantly related paralogue, grouping together with a number of other At-SOC1 paralogues from arabidopsis and other plant species. Gh-SOC1 is inflorescence abundant and no expression was seen in vegetative parts of the plant. Ectopic expression of Gh-SOC1 did not promote flowering, but disturbed the development of floral organs. The epidermal cells of ray flower petals appeared shorter and their shape was altered. The colour of ray flower petals differed from that of the wild-type petals by being darker red on the adaxial side and greenish on the abaxial surface. Several protein–protein interactions with other gerbera MADS domain proteins were identified.

Conclusions

The At-SOC1 paralogue in gerbera shows a floral abundant expression pattern. A late petal expression might indicate a role in the final stages of flower development. Over-expression of Gh-SOC1 led to partial loss of floral identity, but did not affect flowering time. Lines where Gh-SOC1 was downregulated did not show a phenotype. Several gerbera MADS domain proteins interacted with Gh-SOC1.     

Patterns of cell division and expansion in developing petals of Petunia hybrida

The definition of the patterns of cell division and expansion in plant development is of fundamental importance in understanding the mechanics of morphogenesis. By studying cell division and expansion patterns, we have assembled a developmental map of Petunia hybrida petals. Cycling cells were labelled with in situ markers of the cell cycle, whereas cell expansion was followed by assessing cell size in representative regions of developing petals. The outlined cell division and expansion patterns were related to organ asymmetry. Initially, cell divisions are uniformly distributed throughout the petal and decline gradually, starting from the basal part, to form a striking gradient of acropetal polarity. Cell areas, in contrast, increased first in the basal portion and then gradually towards the petal tip. This growth strategy highlighted a cell size control model based on cell-cycle departure time. The dorso-ventral asymmetry can be explained in terms of differential regulation of cell expansion. Cells of the abaxial epidermis enlarged earlier to a higher final extent than those of the adaxial epidermis. Epidermal appendage differentiation contributed to the remaining asymmetry. On the whole our study provides a sound basis for mutant analyses and to investigate the impact of specific (environmental) factors on petal growth.     

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10–100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72–120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10–100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation. Received: 1 December 1997 / Revision received: 13 February 1998 / Accepted: 24 April 1998     

Selective detection of crystalline cellulose in plant cell walls with sum-frequency-generation (SFG) vibration spectroscopy

The selective detection of crystalline cellulose in biomass was demonstrated with sum-frequency-generation (SFG) vibration spectroscopy. SFG is a second-order nonlinear optical response from a system where the optical centrosymmetry is broken. In secondary plant cell walls that contain mostly cellulose, hemicellulose, and lignin with varying concentrations, only certain vibration modes in the crystalline cellulose structure can meet the noninversion symmetry requirements. Thus, SFG can be used to detect and analyze crystalline cellulose selectively in lignocellulosic biomass without extraction of noncellulosic species from biomass or deconvolution of amorphous spectra. The selective detection of crystalline cellulose in lignocellulosic biomass is not readily achievable with other techniques such as XRD, solid-state NMR, IR, and Raman analyses. Therefore, the SFG analysis presents a unique opportunity to reveal the cellulose crystalline structure in lignocellulosic biomass.     

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.     

Determination of copy number and linkage relationships among five actin gene subfamilies in Petunia hybrida

The actin gene superfamily of Petunia hybrida cv. Mitchell contains greater than 100 gene members which have been divided into several highly divergent subfamilies [1]. Five subfamily-specific probes have been used to compare the actin genes among the Mitchell, Violet 23 (V23) and Red 51 (R51) cultivars of P. hybrida. The sum total of actin genes in these five subfamilies was estimated to be between 10 and 34 members in both V23 and R51. Restriction fragment length polymorphisms (RFLPs) between V23 and R51 were examined with these five probes and eleven different restriction endonucleases. Among the 55 comparisons, 87% exhibited RFLPs. These data indicate extreme divergence between V23 and R51 in DNA sequence and/or the presence of small insertions and deletions surrounding these actin gene subfamilies. This divergence suggests that V23 and R51, which have contrasting phenotypic marker loci on every chromosome, may be useful for the development of a complete RFLP linkage map of the Petunia genome. The segregation of Hind III RFLPs among the progeny of two backcrosses demonstrated that representatives of the five subfamilies of Petunia actin genes exist at four distinct genetic locations and suggested that two of these loci are tightly linked. Apparently, amplification of the numerous members of the Petunia actin gene superfamily occurred via gene dispersal of the original subfamily progenitors and not primarily as a result of amplification of a single chromosomal region.     

Ectopic expression of a single homeotic gene, the Petunia gene green petal, is sufficient to convert sepals to petaloid organs.

Genetic studies in Arabidopsis and Antirrhinum showed that petal determination requires the concomitant expression of two homeotic functions, A and B, whereas the A function alone determines sepal identity. The B function is represented by at least two genes. The Petunia homeotic gene green petal (gp) is essential for petal determination as demonstrated by a Petunia gp mutant that has sepals instead of petals. We have used ectopic expression of the gp gene as a tool to study flower development in Petunia. CaMV 35S-gp expression leads to homeotic conversion of sepals into petaloid organs when expressed early in development. This demonstrates that a single homeotic gene is sufficient to induce homeotic conversion of sepals to petals, suggesting that other petal determining genes are regulated in part by ectopically expressed gp. Indeed, two other MADS-box-containing genes, pmads 2 and fbp 1, which show homology to the Antirrhinum B function gene globosa, are activated in the converted petal tissue. Furthermore, our data provide evidence for autoregulation of gp expression in the petaloid tissue and uncover the role of gp in fusion of petal tissues.     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida

? Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. ? PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. ? The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. ? These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.     

Roles of ethylene and 1-aminocyclopropane-1-carboxylic acid in pollination and wound-induced senescence of Petunia hybrida flowers

Normal senescence of Petunia hybrida L. (cv. Pink Cascade) was associated with a 10-fold increase in their ethylene production. Soon after pollination wounding of the stigma of detached flowers there was a burst of ethylene production by the gynoecium, which reached a maximum after 3 h. A subsequnt more gradual rise in ethylene production by the flowers was accompanied by blueing, wilting, and senescence of the corolla. Treatment with 1 μl ethylene 1⁻¹ accelerated the onset of senescence as measured first by color change and then by wilting of the corolla. These changes were further accelerated by using older flowers or higher concentrations of ethylene. Senescence was also hastened by supplying 1-aminocyclopropane-1-carboxylic acid (ACC) through the flower pedicel. Petunia pollen contained high concentrations of ACC (300 nmol g⁻¹); treatment of stigmas with ACC (1 m M ) caused a 4-fold increase in their ethylene production. Senescence, whether natural or hastened by pollination or piercing, was delayed by treating the flowers with the anionic silver thiosulfate complex.     

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 Is Involved in the Production of Negative and Positive Branching Signals in Petunia

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.The dynamic process that leads to a plant''s architecture is regulated by developmental factors and by environmental conditions. Whether or not axillary meristems grow to form branches is one key component of plant architecture. Plants with altered architecture have been important in agronomy since the earliest selections were made by humans. More recent examples are vital to the productivity of our current farming systems. The domestication of maize (Zea mays) and the dwarfing of wheat (Triticum aestivum) and rice (Oryza sativa; as part of the Green Revolution) involved alterations to plant height and branch number that dramatically improved productivity (for review, see Sakamoto and Matsuoka, 2004).Arabidopsis (Arabidopsis thaliana), rice, pea (Pisum sativum), and petunia (Petunia hybrida) are important model plants in which axillary branching has been studied. The growth habits of these plants show differences when grown under standard floral inductive conditions. This is due, in part, to the differing developmental programs controlling the outgrowth of axillary branches. Petunia (inbred genetic stock V26) produces basal axillary branches between nodes two and eight that begin their growth during the vegetative growth phase (Snowden and Napoli, 2003). Axillary branches may also form in the nodes immediately below the first flower after the floral transition (Napoli et al., 1999). Arabidopsis generally produces axillary branches after flowering, releasing axillary meristems in the rosette and also from cauline leaves (Hempel and Feldman, 1994). Wild-type, tall pea cultivars such as Parvus are very unlikely to produce basal axillary branches at any stage of growth but do branch at the nodes immediately below the first flower (Stafstrom, 1995). Cultivated rice produces basal axillary branches, called tillers, during vegetative growth. The tillers formed early in plant development will produce panicles (flowering branches), and the remainder will senesce (Hanada, 1993). How these differences in development arise is yet to be understood.Although the overall architecture of plants varies considerably, the genes so far identified that control branching are frequently conserved between species. In particular, two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, appear to be well conserved among the plant species studied. Mutations in these two genes result in increased branching phenotypes in every species studied to date (Sorefan et al., 2003; Booker et al., 2004; Snowden et al., 2005; Zou et al., 2005; Johnson et al., 2006; Arite et al., 2007). One interesting line of enquiry is to consider whether differences in the regulation or activity of these two genes are involved in the diversity of architecture seen in plants.Grafting experiments have provided insight into the control of axillary branching, in particular the discovery that signals move from roots to shoots. In petunia, Arabidopsis, and pea, some of the increased branching mutants (ccd7 and ccd8 mutants in particular) can be reverted to a wild-type phenotype by grafting mutant scions onto wild-type rootstocks (for review, see Drummond et al., 2009). Additionally, ccd8 mutant plant lines have been reverted to the wild type by the insertion of a small piece (approximately 2 mm) of wild-type hypocotyl into the hypocotyls of mutant petunia or by insertion of a small piece of epicotyl into the epicotyl of mutant pea (Napoli, 1996; Foo et al., 2001). In Arabidopsis, the ccd7 mutant has been similarly reverted using hypocotyl interstock grafts (Booker et al., 2004). Together, these results suggest the presence of a mobile branch inhibitor produced in wild-type tissue. However, an observation by Napoli (1996) suggested that decreased apical dominance1 (dad1) mutant roots may also have a branch-inducing effect in certain circumstances. A similar result was observed for pea in Parvus by Foo et al. (2001). The discussion presented by Napoli (1996) did not exclude either a branch-inducing or a branch-suppressing signal, although current models generally only consider the presence of a branch inhibitor, and recent efforts have focused on the identification of inhibitors of branching.Strigolactones have recently been identified as signaling molecules that inhibit axillary branch outgrowth in plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). Strigolactones were previously identified as signal molecules secreted from roots. When arbuscular mycorrhizae detect strigolactones, they undergo a preinfection hyperbranching response that is thought to aid fungal colonization of the roots, frequently leading to improved nutrient uptake by the plant (Akiyama et al., 2005). The seeds of the parasitic plants Orobanche species and Striga species are also induced to germinate upon detection of strigolactones in the soil, resulting in significant yield losses for some crops (Cook et al., 1966; Siame et al., 1993; Yokota et al., 1998). The production of strigolactones in rice and pea has been shown to require the action of both CCD7 and CCD8 (Gomez-Roldan et al., 2008; Umehara et al., 2008). The discovery that strigolactones can alter branching confirmed a new layer of regulatory complexity in the control of branching that has long been hidden beneath the global plant growth regulators of auxin and cytokinin.In this study, we have focused on the role of the CCD7 gene in the control of branching in petunia. We have isolated a petunia CCD7 ortholog (PhCCD7) and show that the increased branching phenotype of the dad3 mutant is caused by a lesion in this gene. The phenotype of the dad3 mutant is less severe than that of the petunia ccd8 mutant (dad1), and the double ccd7ccd8 mutant is shown to be additive. These observations are contrasted with what has been observed for other plant species. We show that the regulation of PhCCD7 is similar to that of the PhCCD8 gene, with expression predominantly in root and stem tissue (although at a reduced level) and up-regulation of expression in plants with increased numbers of branches. We also provide evidence for the presence of a branch-promoting signal in mutant roots of petunia. These results suggest that there is an added layer of complexity to the control of branching that is not fully described by current models and indicate that the CCD7 gene may have a role in the diversity of plant architecture.