Radial or Bilateral? The Molecular Basis of Floral Symmetry.

In the plant kingdom, the flower is one of the most relevant evolutionary novelties. Floral symmetry has evolved multiple times from the ancestral condition of radial to bilateral symmetry. During evolution, several transcription factors have been recruited by the different developmental pathways in relation to the increase of plant complexity. The MYB proteins are among the most ancient plant transcription factor families and are implicated in different metabolic and developmental processes. In the model plant Antirrhinum majus, three MYB transcription factors (DIVARICATA, DRIF, and RADIALIS) have a pivotal function in the establishment of floral dorsoventral asymmetry. Here, we present an updated report of the role of the DIV, DRIF, and RAD transcription factors in both eudicots and monocots, pointing out their functional changes during plant evolution. In addition, we discuss the molecular models of the establishment of flower symmetry in different flowering plants.

Shared Mutations in a Novel Glutaredoxin Repressor of Multicellular Trichome Fate Underlie Parallel Evolution of Antirrhinum Species.

Most angiosperms produce trichomes-epidermal hairs that have protective or more specialized roles. Trichomes are multicellular in almost all species and, in the majority, secretory. Despite the importance of multicellular trichomes for plant protection and as a source of high-value products, the mechanisms that control their development are only poorly understood. Here, we investigate the control of multicellular trichome patterns using natural variation within the genus Antirrhinum (snapdragons), which has evolved hairy alpine-adapted species or lowland species with a restricted trichome pattern multiple times in parallel. We find that a single gene, Hairy (H), which is needed to repress trichome fate, underlies variation in trichome patterns between all Antirrhinum species except one. We show that H encodes a novel epidermis-specific glutaredoxin and that the pattern of trichome distribution within individuals reflects the location of H expression. Phylogenetic and functional tests suggest that H gained its trichome-repressing role late in the history of eudicots and that the ancestral Antirrhinum had an active H gene and restricted trichome distribution. Loss of H function was involved in an early divergence of alpine and lowland Antirrhinum lineages, and the alleles underlying this split were later reused in parallel evolution of alpines from lowland ancestors, and vice versa. We also find evidence for an evolutionary reversal from a widespread to restricted trichome distribution involving a suppressor mutation and for a pleiotropic effect of H on plant growth that might constrain the evolution of trichome pattern.

The Snapdragon LATE ELONGATED HYPOCOTYL Plays A Dual Role in Activating Floral Growth and Scent Emission.

The plant circadian clock controls a large number of internal processes, including growth and metabolism. Scent emission displays a circadian pattern in many species such as the snapdragon. Here we show that knocking down LATE ELONGATED HYPOCOTYL in Antirrhinum majus affects growth and scent emission. In order to gain an understanding of the growth kinetics, we took a phenomic approach using in-house artificial vision systems, obtaining time-lapse videos. Wild type flowers showed a higher growth speed than knockdown plants. The maximal growth rate was decreased by 22% in plants with lower LHY expression. Floral volatiles were differentially affected as RNAi plants showed advanced emission of compounds synthesized from cinnamic acid and delayed emission of metabolites of benzoic acid. The monoterpenes myrcene and ocimene were delayed, whereas the sesquiterpene farnesene was advanced. Overall, transgenic lines showed an altered volatile emission pattern and displayed a modified scent profile. Our results show that AmLHY plays an important role in the quantitative and qualitative control of floral growth and scent emission.

Assessing Global DNA Methylation Changes Associated with Plasticity in Seven Highly Inbred Lines of Snapdragon Plants (Antirrhinum majus).

Genetic and epigenetic variations are commonly known to underlie phenotypic plastic responses to environmental cues. However, the role of epigenetic variation in plastic responses harboring ecological significance in nature remains to be assessed. The shade avoidance response (SAR) of plants is one of the most prevalent examples of phenotypic plasticity. It is a phenotypic syndrome including stem elongation and multiple other traits. Its ecological significance is widely acknowledged, and it can be adaptive in the presence of competition for light. Underlying genes and pathways were identified, but evidence for its epigenetic basis remains scarce. We used a proven and accessible approach at the population level and compared global DNA methylation between plants exposed to regular light and three different magnitudes of shade in seven highly inbred lines of snapdragon plants (Antirrhinum majus) grown in a greenhouse. Our results brought evidence of a strong SAR syndrome for which magnitude did not vary between lines. They also brought evidence that its magnitude was not associated with the global DNA methylation percentage for five of the six traits under study. The magnitude of stem elongation was significantly associated with global DNA demethylation. We discuss the limits of this approach and why caution must be taken with such results. In-depth approaches at the DNA sequence level will be necessary to better understand the molecular basis of the SAR syndrome.

Spectral effects of light-emitting diodes on plant growth, visual color quality, and photosynthetic photon efficacy: White versus blue plus red radiation.

Arrays of blue (B, 400-500 nm) and red (R, 600-700 nm) light-emitting diodes (LEDs) used for plant growth applications make visual assessment of plants difficult compared to a broad (white, W) spectrum. Although W LEDs are sometimes used in horticultural lighting fixtures, little research has been published using them for sole-source lighting. We grew seedlings of begonia (Begonia ×semperflorens), geranium (Pelargonium ×horturum), petunia (Petunia ×hybrida), and snapdragon (Antirrhinum majus) at 20°C under six sole-source LED lighting treatments with a photosynthetic photon flux density (PPFD) of 160 µmol∙m-2∙s-1 using B (peak = 447 nm), green (G, peak = 531 nm), R (peak = 660 nm), and/or mint W (MW, peak = 558 nm) LEDs that emitted 15% B, 59% G, and 26% R plus 6 µmol∙m-2∙s-1 of far-red radiation. The lighting treatments (with percentage from each LED in subscript) were MW100, MW75R25, MW45R55, MW25R75, B15R85, and B20G40R40. At the transplant stage, total leaf area, and fresh and dry weight were similar among treatments in all species. Surprisingly, when petunia seedlings were grown longer (beyond the transplant stage) under sole-source lighting treatments, the primary stem elongated and had flower buds earlier under MW100 and MW75R25 compared to under B15R85. The color rendering index of MW75R25 and MW45R55 were 72, and 77, respectively, which was higher than those of other treatments, which were ≤64. While photosynthetic photon efficacy of B15R85 (2.25 µmol∙J-1) was higher than the W light treatments (1.51-2.13 µmol∙J-1), the dry weight gain per unit electric energy consumption (in g∙kWh-1) of B15R85 was similar to those of MW25R75, MW45R55, and MW75R25 in three species. We conclude that compared to B+R radiation, W radiation had generally similar effects on seedling growth at the same PPFD with similar electric energy consumption, and improved the visual color quality of sole-source lighting.

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species.

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

Genetics of Floral Development (By Christine Fleet).

Summaryplantcell;29/11/tpc.117.tt1117/FIG1F1fig1A basic model for floral organ identity has been developed using model systems such as Arabidopsis thaliana, snapdragon (Antirrhinum majus), and petunia (Petunia hybrida). In this model, different combinations of proteins known as ABCDE proteins, mostly MADS-domain transcription factors, activate the transcription of target genes to specify the identity of each whorl of floral organs. Changes in the regulation or activation of these target genes contribute to the wide variety of floral forms that we see within and across species. In addition, duplications and divergence of these genes in different groups of flowering plants have resulted in differences in gene function and expression patterns, contributing to differences in flower form across species. Posted December 8, 2017.Click HERE to access Teaching Tool Components.

Formation and Shaping of the Antirrhinum Flower through Modulation of the CUP Boundary Gene.

Boundary domain genes, expressed within or around organ primordia, play a key role in the formation, shaping, and subdivision of planar plant organs, such as leaves. However, the role of boundary genes in formation of more elaborate 3D structures, which also derive from organ primordia, remains unclear. Here we analyze the role of the boundary domain gene CUPULIFORMIS (CUP) in formation of the ornate Antirrhinum flower shape. We show that CUP expression becomes cleared from boundary subdomains between petal primordia, most likely contributing to formation of congenitally fused petals (sympetally) and modulation of growth at sinuses. At later stages, CUP is activated by dorsoventral genes in an intermediary region of the corolla. In contrast to its role at organ boundaries, intermediary CUP activity leads to growth promotion rather than repression and formation of the palate, lip, and characteristic folds of the closed Antirrhinum flower. Intermediary expression of CUP homologs is also observed in related sympetalous species, Linaria and Mimulus, suggesting that changes in boundary gene activity have played a key role in the development and evolution of diverse 3D plant shapes.

Comparative transcription analysis of different Antirrhinum phyllotaxy nodes identifies major signal networks involved in vegetative-reproductive transition.

Vegetative-reproductive phase change is an indispensable event which guarantees several aspects of successful meristem behaviour and organ development. Antirrhinum majus undergoes drastic changes of shoot architecture during the phase change, including phyllotactic change and leaf type alteration from opposite decussate to spiral. However, the regulation mechanism in both of phyllotactic morphology changes is still unclear. Here, the Solexa/Illumina RNA-seq high-throughput sequencing was used to evaluate the global changes of transcriptome levels among four node regions during phyllotactic development. More than 86,315,782 high quality reads were sequenced and assembled into 58,509 unigenes. These differentially expressed genes (DEGs) were classified into 118 pathways described in the KEGG database. Based on the heat-map analysis, a large number of DEGs were overwhelmingly distributed in the hormone signal pathway as well as the carbohydrate biosynthesis and metabolism. The quantitative real time (qRT)-PCR results indicated that most of DEGs were highly up-regulated in the swapping regions of phyllotactic morphology. Moreover, transcriptions factors (TFs) with high transcripts were also identified, controlling the phyllotactic morphology by the regulation of hormone and sugar-metabolism signal pathways. A number of DEGs did not align with any databases and might be novel genes involved in the phyllotactic development. These genes will serve as an invaluable genetic resource for understanding the molecular mechanism of the phyllotactic development.

ERAMOSA controls lateral branching in snapdragon.

Plant forms display a wide variety of architectures, depending on the number of lateral branches, internode elongation and phyllotaxy. These are in turn determined by the number, the position and the fate of the Axillary Meristems (AMs). Mutants that affect AM determination during the vegetative phase have been isolated in several model plants. Among these genes, the GRAS transcription factor LATERAL SUPPRESSOR (Ls) plays a pivotal role in AM determination during the vegetative phase. Hereby we characterize the phylogenetic orthologue of Ls in Antirrhinum, ERAMOSA (ERA). Our data supported ERA control of AM formation during both the vegetative and the reproductive phase in snapdragon. A phylogenetic analysis combined with an analysis of the synteny of Ls in several species strongly supported the hypothesis that ERA is a phylogenetic orthologue of Ls, although it plays a broader role. During the reproductive phase ERA promotes the establishment of the stem niche at the bract axis but, after the reproductive transition, it is antagonized by the MADS box transcription factor SQUAMOSA (SQUA). Surprisingly double mutant era squa plants display a squa phenotype developing axillary meristems, which can eventually turn into inflorescences or flowers.

Generation of shape complexity through tissue conflict resolution.

Out-of-plane tissue deformations are key morphogenetic events during plant and animal development that generate 3D shapes, such as flowers or limbs. However, the mechanisms by which spatiotemporal patterns of gene expression modify cellular behaviours to generate such deformations remain to be established. We use the Snapdragon flower as a model system to address this problem. Combining cellular analysis with tissue-level modelling, we show that an orthogonal pattern of growth orientations plays a key role in generating out-of-plane deformations. This growth pattern is most likely oriented by a polarity field, highlighted by PIN1 protein localisation, and is modulated by dorsoventral gene activity. The orthogonal growth pattern interacts with other patterns of differential growth to create tissue conflicts that shape the flower. Similar shape changes can be generated by contraction as well as growth, suggesting tissue conflict resolution provides a flexible morphogenetic mechanism for generating shape diversity in plants and animals.

Meristem maintenance, auxin, jasmonic and abscisic acid pathways as a mechanism for phenotypic plasticity in Antirrhinum majus.

Plants grow under climatic changing conditions that cause modifications in vegetative and reproductive development. The degree of changes in organ development i.e. its phenotypic plasticity seems to be determined by the organ identity and the type of environmental cue. We used intraspecific competition and found that Antirrhinum majus behaves as a decoupled species for lateral organ size and number. Crowding causes decreases in leaf size and increased leaf number whereas floral size is robust and floral number is reduced. Genes involved in shoot apical meristem maintenance like ROA and HIRZ, cell cycle (CYCD3a; CYCD3b, HISTONE H4) or organ polarity (GRAM) were not significantly downregulated under crowding conditions. A transcriptomic analysis of inflorescence meristems showed Gene Ontology enriched pathways upregulated including Jasmonic and Abscisic acid synthesis and or signalling. Genes involved in auxin synthesis such as AmTAR2 and signalling AmANT were not affected by crowding. In contrast, AmJAZ1, AmMYB21, AmOPCL1 and AmABA2 were significantly upregulated. Our work provides a mechanistic working hypothesis where a robust SAM and stable auxin signalling enables a homogeneous floral size while changes in JA and ABA signalling maybe responsible for the decreased leaf size and floral number.

Validation of Aintegumenta as a gene to modify floral size in ornamental plants.

The gene AINTEGUMENTA (AtANT) is an APETALA2 transcription factor in Arabidopsis activating growth downstream of auxin signalling. Lateral organ size is positively correlated with ANT expression in Arabidopsis. We tested the use of AtANT as a tool to modify floral size in two different plants used as model organisms and ornamental crops, Petunia × hybrida and Antirrhinum majus. Petunia plants expressing PhANT RNAi showed a decrease in PhANT expression correlated with smaller petal limbs. In contrast Petunia plants overexpressing AtANT had larger petal limbs. Petal tube length was less affected in down-regulation of PhANT or overexpression of AtANT. Overexpression of AtANT in Antirrhinum caused increased flower size via increased petal limb width and tube length. Down-regulation of PhANT showed an effect on cell size while overexpression of AtANT in Petunia and Antirrhinum caused significant increases in cell expansion that could explain the differences in floral organ size. The endogenous expression levels of PhANT and AmANT tended to be higher in the limb than in the tube in both Antirrhinum and Petunia. AtANT overexpression caused significant AmANT up-regulation in Antirrhinum limbs but not of PhANT in Petunia, indicating differences in the regulatory network. The differential effect of AtANT on limb and tube in Petunia and Antirrhinum correspond to phenotypic differences observed in natural variation in the corresponding genus indicating a relation between the phenotypic space of a genus and the effect of modified ANT levels, validating ANT as a gene to modify floral size.

Bilateral flower symmetry--how, when and why?

Bilateral flower symmetry has evolved multiple times during flowering plant diversification, is associated with specialized pollination, and is hypothesized to have contributed to flowering plant species richness. The genes and genetic interactions that control bilateral symmetry are well understood in the model species Snapdragon (Antirrhinum majus). I review recent insights into the genetic control of symmetry in Snapdragon. I summarize how this foundational genetic work has been integrated with mathematical modeling approaches, which together provided new insights into the control of quantitative aspects of petal shape. Lastly, I review how evolutionary studies, stemming from knowledge of the genetic control of symmetry in Snapdragon flowers, have revealed extensive parallel recruitment of a similar genetic program during repeated evolution of bilateral symmetry.

TEMPRANILLO is a regulator of juvenility in plants.

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of flowering locus T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species.

Growth and cellular patterns in the petal epidermis of Antirrhinum majus: empirical studies.

BACKGROUND AND AIMS: Analysis of cellular patterns in plant organs provides information about the orientation of cell divisions and predominant growth directions. Such an approach was employed in the present study in order to characterize growth of the asymmetrical wild-type dorsal petal and the symmetrical dorsalized petal of the backpetals mutant in Antirrhinum majus. The aims were to determine how growth in an initially symmetrical petal primordium leads to the development of mature petals differing in their symmetry, and to determine how specific cellular patterns in the petal epidermis are formed. METHODS: Cellular patterns in the epidermis in both petal types over consecutive developmental stages were visualized and characterized quantitatively in terms of cell wall orientation and predominant types of four-cell packets. The data obtained were interpreted in terms of principal directions of growth (PDGs). KEY RESULTS: Both petal types grew predominantly along the proximo-distal axis. Anticlinal cell walls in the epidermis exhibited a characteristic fountain-like pattern that was only slightly modified in time. New cell walls were mostly perpendicular to PDG trajectories, but this alignment could change with wall age. CONCLUSIONS: The results indicate that the predominant orientation of cell division planes and the fountain-like cellular pattern observed in both petal types may be related to PDGs. The difference in symmetry between the two petal types arises because PDG trajectories in the field of growth rates (growth field) controlling petal growth undergo gradual redefinition. This redefinition probably takes place in both petal types but only in the wild-type does it eventually lead to asymmetry in the growth field. Two scenarios of how redefinition of PDGs may contribute to this asymmetry are considered.

A subcellular tug of war involving three MYB-like proteins underlies a molecular antagonism in Antirrhinum flower asymmetry.

The establishment of meristematic domains with different transcriptional activity is essential for many developmental processes. The asymmetry of the Antirrhinum majus flower is established by transcription factors with an asymmetric pattern of activity. To understand how this asymmetrical pattern is established, we studied the molecular mechanism through which the dorsal MYB protein RADIALIS (RAD) restricts the activity of the MYB transcription factor DIVARICATA (DIV) to the ventral region of the flower meristem. We show that RAD competes with DIV for binding with other MYB-like proteins, termed DRIF1 and DRIF2 (DIV- and-RAD-interacting-factors). DRIF1 and DIV interact to form a protein complex that binds to the DIV-DNA consensus region, suggesting that the DRIFs act as co-regulators of DIV transcriptional activity. In the presence of RAD, the interaction between DRIF1 and DIV bound to DNA is disrupted. Moreover, the DRIFs are sequestered in the cytoplasm by RAD, thus, preventing or reducing the formation of DRIF-DIV heterodimers in the nuclei. Our results suggest that in the dorsal region of the Antirrhinum flower meristem the dorsal protein RAD antagonises the activity of the ventral identity protein DIV in a subcellular competition for a DRIF protein promoting the establishment of the asymmetric pattern of gene activity in the Antirrhinum flower.

Evaluation of three ornamental plants for phytoremediation of Pb-contamined soil.

Characteristics of accumulation and tolerance of lead (Pb) in Quamolit pennata, Antirrhinum majus L. and Celosia cristata pyramidalis were investigated to identify Pb-accumulating plants. In this study, pot culture experiment was conducted to assess whether these plants are Pb-hyperaccumulators or accumulators. The results indicated that the Pb enrichment factor (concentration in plant/soil) and Pb translocation factor (concentration in shoot/root) of these plants were principally <1 in pot culture and concentration gradient experiments. However, the Pb concentration in Celosia cristata pyramidalis shoots was higher than 1000 mg kg(-1), the threshold concentration for a Pb-hyperaccumulator. Shoot biomass of Celosia cristata pyramidalis had no significantly (p < 0.05) variation compared to the control. Based on these results, only Celosia cristata pyramidalis could be identified as a Pb-accumulator.

Biolistics-based gene silencing in plants using a modified particle inflow gun.

RNA interference (RNAi) is one of the most commonly used techniques for examining the function of genes of interest. In this chapter we present two examples of RNAi that use the particle inflow gun for delivery of the DNA constructs. In one example transient RNAi is used to show the function of an anthocyanin regulatory gene in flower petals. In the second example stably transformed cell cultures are produced with an RNAi construct that results in a change in the anthocyanin hydroxylation pattern.

Developmental changes in the metabolic network of snapdragon flowers.

Evolutionary and reproductive success of angiosperms, the most diverse group of land plants, relies on visual and olfactory cues for pollinator attraction. Previous work has focused on elucidating the developmental regulation of pathways leading to the formation of pollinator-attracting secondary metabolites such as scent compounds and flower pigments. However, to date little is known about how flowers control their entire metabolic network to achieve the highly regulated production of metabolites attracting pollinators. Integrative analysis of transcripts and metabolites in snapdragon sepals and petals over flower development performed in this study revealed a profound developmental remodeling of gene expression and metabolite profiles in petals, but not in sepals. Genes up-regulated during petal development were enriched in functions related to secondary metabolism, fatty acid catabolism, and amino acid transport, whereas down-regulated genes were enriched in processes involved in cell growth, cell wall formation, and fatty acid biosynthesis. The levels of transcripts and metabolites in pathways leading to scent formation were coordinately up-regulated during petal development, implying transcriptional induction of metabolic pathways preceding scent formation. Developmental gene expression patterns in the pathways involved in scent production were different from those of glycolysis and the pentose phosphate pathway, highlighting distinct developmental regulation of secondary metabolism and primary metabolic pathways feeding into it.