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.

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.

Flower specialisation: the occluded corolla of snapdragons (Antirrhinum) exhibits two pollinator niches of large long-tongued bees.

Flower specialisation of angiosperms includes the occluded corollas of snapdragons (Antirrhinum and some relatives), which have been postulated to be one of the most efficient structures to physical limit access to pollinators. The Iberian Peninsula harbours the highest number of species (18 Iberian of the 20 species of Antirrhinum) that potentially share similar pollinator fauna. Crossing experiments with 18 Iberian species from this study and literature revealed a general pattern of self-incompatibility (SI) - failure in this SI system has been also observed in a few plants - which indicates the need for pollinator agents in Antirrhinum pollination. Field surveys in natural conditions (304 h) found flower visitation (>85%) almost exclusively by 11 species of bee (Anthophora fulvitarsis, Anthophora plumipes, Anthidium sticticum, Apis mellifera, Bombus hortorum, Bombus pascuorum, Bombus ruderatus, Bombus terrestris, Chalicodoma lefebvrei, Chalicodoma pyrenaica and Xylocopa violacea). This result covering the majority of Antirrhinum species suggests that large bees of the two long-tongued bee families (Megachilidae, Apidae) are the major pollinators of Antirrhinum. A bipartite modularity analysis revealed two pollinator systems of long-tongued bees: (i) the long-studied system of bumblebees (Bombus spp.) associated with nine primarily northern species of Antirrhinum; and (ii) a newly proposed pollinator system involving other large bees associated with seven species primarily distributed in southern Mediterranean areas.

Is floral morphology a good predictor of floral visitors to Antirrhineae (snapdragons and relatives)?

The association between plants and flower visitors has been historically proposed as a main factor driving the evolutionary change of both flower and pollinator phenotypes. The considerable diversity in floral morphology within the tribe Antirrhineae has been traditionally related to pollinator types. We used empirical data on the flower visitors from 59 Antirrhineae taxa from the literature and our own field surveys, which provide an opportunity to test whether flower phenotypes are reliable predictors of visitors and pollinator niches. The degree of adjustment between eight key floral traits and actual visitors was explored by testing the predictive value of inferred pollinator syndromes (i.e. suites of floral traits that characterise groups of plant species related to pollination). Actual visitors and inferred pollinator niches (categorisation of visitors' association using a modularity algorithm) were also explored using Linear Discriminant Analysis (LDA). The bee pollinator niche is correctly classified for flowers with dull corolla colour, without nectar guides, as the most important predictor. Both predictive value and statistical classification prove useful in classifying Antirrhineae taxa and the bee pollinator niche, mostly as a consequence of the high proportion of genera and taxa with occluded corollas primarily visited by bees. Our predictive approach rendered a high Positive Predictive Value (PPV) of floral traits in the diagnosis of visitors/pollinator niches. In particular, a high PPV was found for bees as both visitors and forming pollinator niches. In addition, LDA showed that four pollinator niches are well defined based on floral traits. The large number of species visited by bees irrespective of pollinator syndromes leads us to hypothesise their generalist pollinator role, despite the phenotypically specialised flowers of Antirrhineae.

Learning about natural variation of odor mixtures enhances categorization in early olfactory processing.

Natural odors are typically mixtures of several chemical components. Mixtures vary in composition among odor objects that have the same meaning. Therefore a central 'categorization' problem for an animal as it makes decisions about odors in natural contexts is to correctly identify odor variants that have the same meaning and avoid variants that have a different meaning. We propose that identified mechanisms of associative and non-associative plasticity in early sensory processing in the insect antennal lobe and mammalian olfactory bulb are central to solving this problem. Accordingly, this plasticity should work to improve categorization of odors that have the opposite meanings in relation to important events. Using synthetic mixtures designed to mimic natural odor variation among flowers, we studied how honey bees learn about and generalize among floral odors associated with food. We behaviorally conditioned honey bees on a difficult odor discrimination problem using synthetic mixtures that mimic natural variation among snapdragon flowers. We then used calcium imaging to measure responses of projection neurons of the antennal lobe, which is the first synaptic relay of olfactory sensory information in the brain, to study how ensembles of projection neurons change as a result of behavioral conditioning. We show how these ensembles become 'tuned' through plasticity to improve categorization of odors that have the different meanings. We argue that this tuning allows more efficient use of the immense coding space of the antennal lobe and olfactory bulb to solve the categorization problem. Our data point to the need for a better understanding of the 'statistics' of the odor space.

The genetic control of flower-pollinator specificity.

The ca. 275,000 species of flowering plants are the result of a recent adaptive radiation driven largely by the coevolution between plants and their animal pollinators. Identification of genes and mutations responsible for floral trait variation underlying pollinator specificity is crucial to understanding how pollinator shifts occur between closely related species. Petunia, Mimulus, and Antirrhinum have provided a high standard of experimental evidence to establish causal links from genes to floral traits to pollinator responses. In all three systems, MYB transcription factors seem to play a prominent role in the diversification of pollinator-associated floral traits.

The influence of pigmentation patterning on bumblebee foraging from flowers of Antirrhinum majus.

Patterns of pigmentation overlying the petal vasculature are common in flowering plants and have been postulated to play a role in pollinator attraction. Previous studies report that such venation patterning is significantly more attractive to bee foragers in the field than ivory or white flowers without veins. To dissect the ways in which venation patterning of pigment can influence bumblebee behaviour, we investigated the response of flower-naïve individuals of Bombus terrestris to veined, ivory and red near-isogenic lines of Antirrhinum majus. We find that red venation shifts flower colour slightly, although the ivory background is the dominant colour. Bees were readily able to discriminate between ivory and veined flowers under differential conditioning but showed no innate preference when presented with a free choice of rewarding ivory and veined flowers. In contrast, both ivory and veined flowers were selected significantly more often than were red flowers. We conclude that advantages conferred by venation patterning might stem from bees learning of their use as nectar guides, rather than from any innate preference for striped flowers.

Patterns of floral colour neighbourhood and their effects on female reproductive success in an Antirrhinum hybrid zone.

The maintenance of genetic integrity of parental populations is often explained by selection against hybrids. However, the selection agents are usually unknown. The role of environmental biotic interactions is often suspected but has rarely been demonstrated. In plants for instance, mutualism with pollinators may be involved. After verification that pollen deposition is a limiting factor for fruit set, we used an individual-based study and a representation of pollinator colour perception to test the effects of local plant density and floral colour neighbourhood on female reproductive success in an Antirrhinum hybrid zone. In addition to flower colour and density effects, the composition of the floral neighbourhood was found to influence fruit set, suggesting that most plants were usually better fertilized when similar to their neighbours. However, the plants of one particular type were sometimes favoured when very different from their neighbours. The implications for hybrid zone dynamics are discussed.

Generation of diverse biological forms through combinatorial interactions between tissue polarity and growth.

A major problem in biology is to understand how complex tissue shapes may arise through growth. In many cases this process involves preferential growth along particular orientations raising the question of how these orientations are specified. One view is that orientations are specified through stresses in the tissue (axiality-based system). Another possibility is that orientations can be specified independently of stresses through molecular signalling (polarity-based system). The axiality-based system has recently been explored through computational modelling. Here we develop and apply a polarity-based system which we call the Growing Polarised Tissue (GPT) framework. Tissue is treated as a continuous material within which regionally expressed factors under genetic control may interact and propagate. Polarity is established by signals that propagate through the tissue and is anchored in regions termed tissue polarity organisers that are also under genetic control. Rates of growth parallel or perpendicular to the local polarity may then be specified through a regulatory network. The resulting growth depends on how specified growth patterns interact within the constraints of mechanically connected tissue. This constraint leads to the emergence of features such as curvature that were not directly specified by the regulatory networks. Resultant growth feeds back to influence spatial arrangements and local orientations of tissue, allowing complex shapes to emerge from simple rules. Moreover, asymmetries may emerge through interactions between polarity fields. We illustrate the value of the GPT-framework for understanding morphogenesis by applying it to a growing Snapdragon flower and indicate how the underlying hypotheses may be tested by computational simulation. We propose that combinatorial intractions between orientations and rates of growth, which are a key feature of polarity-based systems, have been exploited during evolution to generate a range of observed biological shapes.

Determining the contribution of epidermal cell shape to petal wettability using isogenic Antirrhinum lines.

The petal epidermis acts not only as a barrier to the outside world but also as a point of interaction between the flower and potential pollinators. The presence of conical petal epidermal cells has previously been shown to influence the attractiveness of the flower to pollinating insects. Using Antirrhinum isogenic lines differing only in the presence of a single epidermal structure, conical cells, we were able to investigate how the structure of the epidermis influences petal wettability by measuring the surface contact angle of water drops. Conical cells have a significant impact on how water is retained on the flower surface, which may have indirect consequences for pollinator behaviour. We discuss how the petal epidermis is a highly multifunctional one and how a battery of methods, including the use of isogenic lines, is required to untangle the impacts of specific epidermal properties in an ecological context.

A comparison of antirrhinoside distribution in the organs of two related Plantaginaceae species with different reproductive strategies.

A study of two related plants (Antirrhinum majus L. and Linaria vulgaris Mill.) containing the same defensive compound (the iridoid glucoside, antirrhinoside) but with reproductive strategies that differ during ontogeny was undertaken. Young leaves are important to plants due to their higher photosynthetic rates and, therefore, should be better protected with higher concentrations of defensive compounds such as antirrhinoside. Declining concentrations of antirrhinoside as leaves aged was found for A. majus but this was generally not the case for L. vulgaris. Concentrations of antirrhinoside in root tissue were low and constant throughout ontogeny for A. majus whereas for L. vulgaris root levels of antirrhinoside were high during the period when vegetative growth is its sole means of reproduction. Antirrhinoside in L. vulgaris roots declined relative to A. majus roots during budding and flowering. During flowering, significantly less antirrhinoside and relative biomass are devoted to L. vulgaris flowers than in A. majus. While these findings are consistent with Optimal Defense Theory (ODT) further work on the distribution of antirrhinoside and the effect of insect herbivory on plant fitness in other related species is needed.

Multiple regulatory mechanisms influence the activity of the transposon, Tam3, of Antirrhinum.

In Antirrhinum, several unique regulations of the transposon, Tam3, have been described. Tam3 activity in Antirrhinum is strictly controlled by the growing temperature of plants (low-temperature-dependent transposition: LTDT), by chromosomal position of Tam3 copy and by two specific repressor genes Stabiliser (St) and New Stabiliser (NSt). Here, the effects of the St and NSt loci on Tam3 transposition are compared. In cotyledons and hypocotyls, Tam3 is active even at high growing temperatures, indicating that LTDT does not operate when these organs are developing. This developmental regulation of Tam3 activity is differentially influenced by the St and NSt loci: St permits Tam3 transposition in cotyledons and hypocotyls, whereas NSt suppresses it in these organs. The effects of these host genes on Tam3 activity at the molecular level were examined. It was found that neither of these genes inhibits the transcription of the Tam3 transposase gene nor its translation, and that the Tam3 transposase has the potential to catalyze transposition in the St and NSt lines. The differences between the effects of St and NSt imply that they regulate Tam3 activity independently. Our molecular data indicate that their influence on Tam3 transposition seems to be nonepigenetic; possible mechanisms for their activity are discussed.

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes.

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.

Control of cell and petal morphogenesis by R2R3 MYB transcription factors.

Petals of animal-pollinated angiosperms have adapted to attract pollinators. Factors influencing pollinator attention include colour and overall size of flowers. Colour is determined by the nature of the pigments, their environment and by the morphology of the petal epidermal cells. Most angiosperms have conical epidermal cells, which enhance the colour intensity and brightness of petal surfaces. The MYB-related transcription factor MIXTA controls the development of conical epidermal cells in petals of Antirrhinum majus. Another gene encoding an R2R3 MYB factor very closely related to MIXTA, AmMYBML2, is also expressed in flowers of A. majus. We have analysed the roles of AmMYBML2 and two MIXTA-related genes, PhMYB1 from Petunia hybrida and AtMYB16 from Arabidopsis thaliana, in petal development. The structural similarity between these genes, their comparable expression patterns and the similarity of the phenotypes they induce when ectopically expressed in tobacco, suggest they share homologous functions closely related to, but distinct from, that of MIXTA. Detailed phenotypic analysis of a phmyb1 mutant confirmed the role of PhMYB1 in the control of cell morphogenesis in the petal epidermis. The phmyb1 mutant showed that epidermal cell shape affects petal presentation, a phenotypic trait also observed following re-examination of mixta mutants. This suggests that the activity of MIXTA-like genes also contributes to petal form, another important factor influencing pollinator attraction.

Genetic diversity and the reproductive system in related species of antirrhinum.

BACKGROUND AND AIMS: Seven related species of Antirrhinum (A. siculum, A. majus, A. latifolium, A. linkianum, A. litigiosum, A. cirrhigherum and A. tortuosum) were studied in order to compare levels of genetic variation and its partitioning in them, and to check relationships between genetic patterns and the reproductive system. METHODS: Eight hundred and fifty-one plants were screened for variability at 13 allozyme loci by means of horizontal starch gel electrophoresis. Parameters of genetic diversity and its partitioning, the inbreeding coefficient as well as an indirect estimate of gene flow based on the equation: Nm = (1 - G(ST))/4G(ST), were calculated. KEY RESULTS: Genetic variability in A. siculum was found to be the lowest known in the genus. Mean values of F(IT) and F(IS) were mostly positive and not significantly different from zero. Population differentiation (F(ST)) ranged between 6.1 in A. tortuosum and 17.6 in A. linkianum. The inbreeding coefficient within populations ranged between F(IS) = -0.5 in A. tortuosum and F(IS) = 1 in A. siculum. Estimates of gene flow ranged between Nm = 15 in A. majus (considered as very high) to Nm = 0.42 in A. siculum (considered as low). CONCLUSIONS: Correlation was found between levels of diversity and differentiation on one hand, and the reproductive system of the studied taxa on the other. Striking differences among species in the inbreeding coefficient (F(IS)) show different reproductive systems, which mostly support previous reports. Strategies for the conservation of A. siculum are recommended, such as preservation of natural populations as well as ex situ preservation of seeds from different populations.

A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum.

The Rosea1, Rosea2, and Venosa genes encode MYB-related transcription factors active in the flowers of Antirrhinum majus. Analysis of mutant phenotypes shows that these genes control the intensity and pattern of magenta anthocyanin pigmentation in flowers. Despite the structural similarity of these regulatory proteins, they influence the expression of target genes encoding the enzymes of anthocyanin biosynthesis with different specificities. Consequently, they are not equivalent biochemically in their activities. Different species of the genus Antirrhinum, native to Spain and Portugal, show striking differences in their patterns and intensities of floral pigmentation. Differences in anthocyanin pigmentation between at least six species are attributable to variations in the activity of the Rosea and Venosa loci. Set in the context of our understanding of the regulation of anthocyanin production in other genera, the activity of MYB-related genes is probably a primary cause of natural variation in anthocyanin pigmentation in plants.

Evolution of regulatory interactions controlling floral asymmetry.

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.

Evolution in action: following function in duplicated floral homeotic genes.

Gene duplication plays a fundamental role in evolution by providing the genetic material from which novel functions can arise. Newly duplicated genes can be maintained by subfunctionalization (the duplicated genes perform different aspects of the original gene's function) and/or neofunctionalization (one of the genes acquires a novel function). PLENA in Antirrhinum and AGAMOUS in Arabidopsis are the canonical C-function genes that are essential for the specification of reproductive organs. These functionally equivalent genes encode closely related homeotic MADS-box transcription factors. Using genome synteny, we confirm phylogenetic analyses showing that PLENA and AGAMOUS are nonorthologous genes derived from a duplication in a common ancestor. Their respective orthologs, SHATTERPROOF in Arabidopsis and FARINELLI in Antirrhinum, have undergone independent subfunctionalization via changes in regulation and protein function. Surprisingly, the functional divergence between PLENA and FARINELLI, is morphologically manifest in both transgenic Antirrhinum and Arabidopsis. This provides a clear illustration of a random evolutionary trajectory for gene functions after a duplication event. Different members of a duplicated gene pair have retained the primary homeotic functions in different lineages, illustrating the role of chance in evolution. The differential ability of the Antirrhinum genes to promote male or female development provides a striking example of subfunctionalization at the protein level.

INCOMPOSITA: a MADS-box gene controlling prophyll development and floral meristem identity in Antirrhinum.

INCOMPOSITA (INCO) is a MADS-box transcription factor and member of the functionally diverse StMADS11 clade of the MADS-box family. The most conspicuous feature of inco mutant flowers are prophylls initiated prior to first whorl sepals at lateral positions of the flower primordium. The developing prophylls physically interfere with subsequent floral organ development that results in aberrant floral architecture. INCO, which is controlled by SQUAMOSA, prevents prophyll formation in the wild type, a role that is novel among MADS-box proteins, and we discuss evolutionary implications of this function. Overexpression of INCO or SVP, a structurally related Arabidopsis MADS-box gene involved in the negative control of Arabidopsis flowering time, conditions delayed flowering in transgenic plants, suggesting that SVP and INCO have functions in common. Enhanced flowering of squamosa mutants in the inco mutant background corroborates this potential role of INCO as a floral repressor in Antirrhinum. One further, hitherto hidden, role of INCO is the positive control of Antirrhinum floral meristem identity. This is revealed by genetic interactions between inco and mutants of FLORICAULA, a gene that controls the inflorescence to floral transition, together with SQUAMOSA. The complex regulatory and combinatorial relations between INCO, FLORICAULA and SQUAMOSA are summarised in a model that integrates observations from molecular studies as well as analyses of expression patterns and genetic interactions.

Molecular and genetic interactions between STYLOSA and GRAMINIFOLIA in the control of Antirrhinum vegetative and reproductive development.

STYLOSA (STY) in Antirrhinum and LEUNIG (LUG) in Arabidopsis control the spatially correct expression of homeotic functions involved in the control of floral organ identity. We show here that the sty mutant also displays alteration in leaf venation patterns and hypersensitivity towards auxin and polar auxin transport inhibitors, demonstrating that STY has a more general role in plant development. STY and LUG are shown to be orthologues that encode proteins with structural relation to GRO/TUP1-like co-repressors. Using a yeast-based screen we found that STY interacts with several transcription factors, suggesting that STY, like GRO/TUP1, forms complexes in vivo. Proteins of the YABBY family, characterised by containing a partial HMG domain, represent a major group of such interactors. In vivo association of STY with one of the YABBY proteins, GRAMINIFOLIA (GRAM), is supported by enhanced phenotypic defects in sty gram double mutants, for instance in the control of phyllotaxis, floral homeotic functions and organ polarity. Accordingly, the STY and GRAM protein and mRNA expression patterns overlap in emerging lateral organ primordia. STY is expressed in all meristems and later becomes confined to the adaxial domain and (pro)vascular tissue. This pattern is similar to genes that promote adaxial identity, and, indeed, STY expression follows, although does not control, adaxial fate. We discuss the complex roles of STY and GRAM proteins in reproductive and vegetative development, performed in part in physical association but also independently.