Extrafloral Nectaries of Dioscorea rotundata Poir.: Their Structure and Secretions

Extrafloral nectaries are to be found embedded in the leaf laminaof Dioscorea rotundata Poir., with a pore opening on to thelower leaf surface. The nectaries comprise small, densely cytoplasmiccells and are bounded by a layer of cells containing littleor no cytoplasm. Their secretion contains sucrose, fructoseand glucose with traces of galactose. Ninhydrin-positive compoundsare also present. Diosorra rotunrdata Poir, extrafloral nectaries, secretion, ultrastructure     

Foliar Nectaries and Glandular Trichomes in Catalpa (Bignoniaceae)

The Tertiary relic genus Catalpa, containing 11 Old and New World species, has multiple nectaries in the junctions of the major veins on the lower leaf surfaces. The generalized structure of the nectaries consists of a single, basal cell, and a single row of vertically oriented secretory cells. The nectaries are small, and contain no vascular tissue. Glandular and nonglandular trichomes are also present on the leaves. The glandular trichomes are structurally very similar to the nectaries, and probably are their precursors. Development of multiple nectaries in the lower leaf surface vein axils is considered to be an advanced strategy for attracting beneficial insects to control or minimize the effects of herbivorous insects. Chinese and American species of Catalpa are closely related, and have greater numbers of nectaries in more locations on the lower leaf surface than west Indian species. Herbivory pressures in the West Indies are postulated to be lower than on the continents, i.e. Asia and North America.     

THE FUNCTION OF EXTRAFLORAL NECTARIES IN OPUNTIA ACANTHOCARPA (CACTACEAE)

Opuntia acanthocarpa (Cactaceae) possesses extrafloral nectaries embedded in the areoles of new reproductive and vegetative growth. The nectar secreted by these glands attracts ants and is a nutritional food source. Members of one attracted ant species, Crematogaster opuntiae (Myrmicinae), are aggressive and efficient defenders of the plants against cactus-feeding insects. The results of our study are consistent with the ant-guard hypothesis for the role of extrafloral nectaries in O. acanthocarpa. Additionally, individuals of O. acanthocarpa are well protected in comparison with those of O. phaeacantha. The latter generally possess ephemeral extrafloral nectaries and consistently maintain fewer ants.     

DISTRIBUTION OF DEFENSE NECTARIES IN IPOMOEA (CONVOLVULACEAE)

The structure and distribution of defense nectaries in the genus Ipomoea (Convolvulaceae) were investigated. These nectaries do not reward pollinators and probably contribute to antiherbivore defense. Of 22 species sectioned, 15 had defense nectaries on the sepals. Of 12 other species observed, ten had sepal nectaries and two did not. Structurally, 14 of the species sectioned had crypt sepal nectaries and one had a basin nectary. Of the 14 species with crypt nectaries, two had invaginated spaces adding greatly to the internal area of these nectaries, and forming the most complex nectaries that have been reported. We term these labyrinthine crypt nectaries. All three types of nectaries are lined with secretory trichomes along the proximal surfaces of the crypts. Species with defense nectaries on the sepals tend to have petiolar defense nectaries as well, but the two locations may have different nectary types; e.g., basins on the petiole and crypts on the sepals. Since most reports of the function of these nectaries have shown antiherbivore defense by nectar feeders, the distributions of defense nectaries with respect to region and life history of the species were sought. Plants without sepal nectaries were found to have significantly smaller seeds than plants with sepal nectaries; they were also more frequently annuals. No significant relationship was found between region or breeding system and defense nectaries.     

Extrafloral nectaries in the genus Macaranga (Euphorbiaceae) in Malaysia: comparative studies of their possible significance as predispositions for myrmecophytism

Some species of the paleotropical tree genus Macaranga (Euphorbiaceae) live in close association with ants. The genus comprises the full range of species from those not regularly inhabited by ants to obligate myrmecophytes. In Malaysia (Peninsular and Borneo) 23 of the 52 species are known to be ant-associated (44%). The simplest structural adaptation of plants to attract ants are extrafloral nectaries. We studied the distribution of extrafloral nectaries in the genus Macaranga to assess the significance of this character as a possible predisposition for the evolution of obligate myrmecophytism. All species have marginal glands on the leaves. However, only the glands of non- myrmecophytic species function as nectaries, whereas liquids secreted by these glands in myrmecophytic species did not contain sugar. Some non-myrmecophytic Macaranga and transitional Macaranga species in addition have extrafloral nectaries on the leaf blade near the petiole insertion. All obligatorily myrmecophytic Macaranga species, however, lack additional glands on the lamina. The non-myrmecophytic species are visited by a variety of different ant species, whereas myrmecophytic Macaranga are associated only with one specific ant-partner. Since these ants keep scale insects in the hollow stems, reduction of nectary production in ant-inhabited Macaranga seems to be biologically significant. We interpret this as a means of (a) saving the assimilates and (b) stabilization of maintenance of the association's specificity. Competition with other ant species for food rewards is avoided and thereby danger of weakening the protective function of the obligate ant- partner for the plant is reduced. A comparison with other euphorb species living in the same habitats as Macaranga showed that in genera in which extrafloral nectaries are widespread, no myrmecophytes have evolved. Possession of extrafloral nectaries does not appear to be essential for the development of symbiotic ant-plant interactions. Other predispositions such as nesting space might have played a more important role.     

THE FLORAL AND EXTRA-FLORAL NECTARIES OF PASSIFLORA. II. THE EXTRA-FLORAL NECTARY

Extra-floral nectaries of nine species of Passiflora were studied with light and electron microscopy prior to and during secretion. There is no evidence of ER or Golgi participation in the secretion of nectar. The vascular tissue supplying the nectary is characterized by companion and phloem parenchyma cells which are usually larger than the sieve elements, a configuration similar to that found in leaf minor veins. In the petiolar nectaries, large masses of membrane-bound protein are commonly found in these cells. This protein is absent in laminar nectaries.     

THE EXTRAFLORAL NECTARIES OF IPOMOEA LEPTOPHYLLA (CONVOLVULACEAE)

Ipomoea leptophylla Torr. (Convolvulaceae) is a sprawling dry-site morning glory with two types of extrafloral nectaries: foliar nectaries and nectaries on the outside of the sepals. Both are shown to greatly increase insect visitation to the plant. Ants visiting sepal-surface nectaries significantly decrease flower damage caused by grasshoppers and seed losses caused by bruchids. These results are similar to those for I. carnea and other plants whose extrafloral nectary-ant interactions have been studied, but differ in detail. This is the first demonstration of antiherbivore defense of a prairie plant by nectary visitors.     

Comparative morphology of the leaf epidermis in the genera Codonanthe (Martius) Hanstein and Nematanthus Schrader (Gesneriaceae)*

The leaf epidermis of 14 species ofCodonanthe and 10 species of Nematanthus has been examined. Species of Codonanthe section Codonanthe are geographically restricted to south-eastern Brazil, and are diploid. They possess multicellular-uniseriate nonglandular trichomes, glandular trichomes with a four-celled head and a short body, anisocytic stomata and lack extrafloral nectaries. Species of Codonanthe section Spathuliformae and Codonanthe subgenus Codonanthella are distributed from southern Mexico through Central America to north-western South America and are tetraploid. They possess unicellular non-glandular trichomes (except C. caribaea), glandular trichomes with a two-celled head (except C. caribaea) and a short body, anisocytic stomata and extrafloral nectaries (except C. caribaea). All Nematanthus species are distributed in south-eastern Brazil and are diploid (N=8). Six species of Nematanthus consistently have multicellular-uniseriate nonglandular trichomes, glandular trichomes with a four-celled head and a short (unicellular) or long (multicellular) body, anisocytic stomata and lack extrafloral nectaries. Four species of Nematanthus have multicellular-uniseriate non-glandular trichomes, glandular trichomes with a head of more than four cells and a short body, anisocytic and helicocytic stomata and lack extrafloral nectaries.     

Sooty Mould on Hibiscus rosa‐sinensis Caused by Leptoxyphium kurandae is Associated with Extrafloral Nectaries

In August 2013, sooty mould was observed on Chinese hibiscus (Hibiscus rosa‐sinensis) in a propagation nursery in Seoul, Korea. The sooty mould initially developed at the junction between the leaf blade and leaf petiole and then dispersed along the vein on the abaxial surface. The fungal growth pattern on the plants was quite different from general sooty moulds growing on honeydew secreted by insects on the plants. On the basis of the morphological characteristics and phylogenetic analysis using the internal transcribed spacer rDNA, this fungus was identified as Leptoxyphium kurandae. A pathogenicity test was carried out to fulfil Koch's postulates. Through field observation and a pathogenicity test, we found an association between the sooty mould and extrafloral nectaries. To our knowledge, this is the first report of sooty mould caused by L. kurandae on the extrafloral nectaries of H. rosa‐sinensis.     

Pollination-related characteristics in the mimosoid legumeAcacia terminalis (Leguminosae)

Intraspecific variation has been found for several pollination-related characteristics in two isolated populations of the self-incompatible treeAcacia terminalis: floral characteristics including colour and flowering time; style length; size and colour of extrafloral nectaries on the leaf petioles; chemical components of the extrafloral nectar; different taxa of bee pollinators; and frequency differences in bird pollinators. These differences possibly reflect the evolution of two different pollination syndromes within this species.     

Population variation in plant traits associated with ant attraction and herbivory in Chamaecrista fasciculata (Fabaceae)

The benefits of ant–plant–herbivore interactions for the plant depend on the abundance of ants and herbivores and the selective pressures these arthropods exert. In plants bearing extrafloral nectaries (EFN), different mean trait values may be selected for by different populations in response to local herbivore pressure, ultimately resulting in the evolution of differences in plant traits that attract ants as defensive agents against herbivory. To determine if variation in traits that mediate ant–plant interactions reflect herbivore selective pressures, we quantified intra- and inter-population variation in plant traits for eight populations of the EFN-bearing annual Chamaecrista fasciculata (Michx.) (Fabaceae). Censuses in rural and urban areas of Missouri and Illinois (USA) showed population differences in ant attendance and herbivore pressure. Seeds were collected from each population, and plants were grown in a common greenhouse environment to measure sugar production, nectar volume and composition, EFN size and time of emergence, leaf pubescence, and leaf quality throughout plant development. Populations varied mainly in terms of nectary size, sugar production, and nectar volume, but to a lesser degree in leaf pubescence. Populations of C. fasciculata within urban areas (low in insect abundance) had small nectaries and the lowest nectar production. There was a positive correlation across populations between herbivore density and leaf damage by those herbivores on the one hand and sugar production and nectar volume on the other. These results, in conjunction with lack of evidence for maternally based environmental effects, suggest that population differences in herbivore damage have promoted differential evolution of EFN-related traits among populations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

THE EXTRAFLORAL NECTARIES OF IPOMOEA CARNEA (CONVOLVULACEAE)

Ipomoea carnea (Convolvulaceae) possesses two types of extrafloral nectaries, located on the petiole and on the pedicel. These secrete a complex nectar containing sugars and amino acids. The insects attracted to the extrafloral nectaries are predominantly ants and they are relatively abundant throughout the year. A number of incidents of plant defense as a result of the presence of extrafloral nectary visitors at the extrafloral nectaries of I. carnea were observed and are consistent with the ant-guard theory of the function of extrafloral nectaries.     

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

A DEVELOPMENTAL STUDY OF EXTRAFLORAL NECTARIES IN SLIPPER SPURGE (PEDILANTHUS TITHYMALOIDES,EUPHORBIACEAE)

Slipper spurge (Pedilanthus tithymaloides) bears one or two stalked extrafloral nectaries on either side of the leaf base and one at the leaf tip. The mature nectary is differentiated into multicellular zones: head, neck, and stipe. The nectary arises as a small group of meristematic cells with densely staining cytoplasm and nuclei. The columnar secretory cells show changes in their chemical nature at different developmental stages of the nectary. There is a basipetal sequence in the development and decay of the tissues in the nectary. Decay of the nectary begins at the head, abscission occurs at the line between stipe and neck regions, and the scars of the fallen nectaries are left when the stipe cells also collapse.     

Stipular extranuptial nectaries new to Polygala: morphology and ontogeny

Although taxonomic studies indicate that approximately one‐third of the genera of Polygalaceae have nodal glands, few anatomical data are available on the structure and ontogeny of these secretory organs. We studied the as yet unknown origin, structure and function of such glands in Polygala laureola. During field observations, we detected glucose in the secretion using Glicofita Plus® and visitors were recorded. Vegetative shoot apices and nodal glands were examined using light microscopy and scanning electron microscopy. The presence of glucose in the secretion allowed us to identify these nodal glands as extranuptial nectaries. Secretory cells occupy a medullary position and are surrounded by phloem. Vascular bundles are concentric, and xylem is only observed at the basal region of the nectary. Nectar is released during the daytime through a pore at the top of the nectary. A stipular origin was confirmed by the fact that the procambial strand is connected to the leaf trace, opposite the leaf gap. The occurrence of stipular extranuptial nectaries in a nodal position is new to Polygala. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166 , 40–50.     

PROTEIN-CONTAINING CELLS IN THE NECTARY PHLOEM OF PASSIFLORA WARMINGII

Passiflora warmingii petiolar nectaries are characterized by the presence of large protein-containing phloem parenchyma cells which occupy the bulk of the nectary. Immature, mature, and senescent nectaries, as well as stem tips and petioles from unexpanded and mature leaves, were studied to learn the origin and fate of the protein and to determine if similar protein-containing cells occur in main-path phloem. The protein is present as membrane-limited fibrils in the phloem parenchyma of immature nectaries and in young main-path phloem. In the nectary, it persists until leaf senescence but becomes highly dispersed and barely detectable in mature main-path phloem parenchyma. Although superficially resembling P-protein it is always surrounded by a membrane, has smaller dimensions than is reported for P-protein, appears to be derived from RER, and is found in association with typical P-protein in the same cell. Possible functions for this material are suggested.     

Distribution and structural aspects of extrafloral nectaries in Cedrela fissilis (Meliaceae)

The occurrence of extrafloral nectaries (EFNs) in Meliaceae has been reported for some genera, but little anatomical data are available. Therefore, to determine the distribution and structural aspects of EFNs, Cedrela fissilis Vell. leaves in different stages of development were collected, fixed, and processed for light and scanning electron microscopy. On the petiole, rachis and petiolule, EFNs were found to be arranged predominantly towards the abaxial surface, while their occurrence in leaflet blades was restricted to the abaxial surface of the major veins, noticeably on the midrib. Basal leaflets displayed few EFNs; however, we observed an increase towards the leaf's apex. The leaf can contain more than 300 inconspicuous EFNs, which show secretory activity throughout the leaf's life. Two EFN morphotypes were visible: flattened or elevated, both circular or slightly elliptical and similar in origin and tissue composition. The secretory tissue is embedded in the rachis cortex or in the major veins of the leaf blade and EFNs are not vascularized. The EFN secretory pole shows a uniseriate epidermis with compactly arranged cells and a thin cuticle; stomata and trichomes are absent. The observation of ant visits at these structures reinforces the assumption that EFNs mediate ant–plant interactions and play a protective role against herbivores throughout the life of a leaf.     

Unraveling the secretion mechanism of the curious nectaries in Gentianaceae

Unusual nectaries were anatomically described as being usual traits for Gentianaceae. They are small, avascularized, and formed by 3 to 5 rosette cells with labyrinthine walls around one central cell. Such as nectaries have been reported for stems, petals, and sepals of different species of the family, however, there is no information on the mechanisms involved with the synthesis and release of secretion. Thus, this work aimed to unravel the mechanism of secretion and exudation of nectar for these curious nectaries using Calolisianthus speciosus as a model. Samples were processed according to standard methods for light and electron microscopy. Leaf and sepal nectaries were described, as were those of the apex of petals where ants were observed patrolling a darkened region. The enzymatic method was used for the detection of sugars, proteins, and amino acids in leaf and sepal exudates. The nectaries of petals of C. speciosus are similar to those of its leaves, sepals, and stem, although their activities are asynchronous. Polysaccharides were detected on the labyrinthine walls of rosette cells and protein in the opposite region of the cytoplasm. Labyrinthine walls increase the contact surface between rosette cells and the central cell, allowing for the transfer of secretion. After accumulation, the secretion is released to the subcuticular space of the central cell through disruption of the cuticle. The secretion and exudation of nectar were elucidated and involve distinct organelles.

     

Two Mallotus species of different life histories adopt different defense strategies in relation to leaf age

Plants defend their leaves using multiple defense traits that change functions with leaf age. We examined the effects of leaf age on the development of multiple defense traits in two related Mallotus (Euphorbiaceae) species: young plants of the fast‐growing Mallotus japonicus (Spreng.) Müll. Arg. and the slow‐growing Mallotus philippensis (Lam.) Müll. Arg. Sequential leaves of the two species were measured for their leaf area, leaf mass/area, densities of trichomes and pellucid dots, extrafloral nectar volume, and the numbers of extrafloral nectaries and pearl bodies. Mallotus japonicus shifted its defense tactics from direct defense using trichomes and pellucid dots in young leaves to biotic defense using extrafloral nectar and pearl bodies in middle‐aged leaves. In contrast, M. philippensis used direct, chemical defense throughout all leaf ages, together with the shift from indirect, biotic defense using extrafloral nectar in young leaves to direct, physical defense using leaf toughness in middle‐aged leaves. These results strongly suggest that, in relation to life history, plants can alter optimal combinations of multiple defense traits with leaf age.     

Genetic and evolution analysis of extrafloral nectary in cotton

Extrafloral nectaries are a defence trait that plays important roles in plant–animal interactions. Gossypium species are characterized by cellular grooves in leaf midribs that secret large amounts of nectar. Here, with a panel of 215 G. arboreum accessions, we compared extrafloral nectaries to nectariless accessions to identify a region of Chr12 that showed strong differentiation and overlapped with signals from GWAS of nectaries. Fine mapping of an F₂ population identified GaNEC1, encoding a PB1 domain‐containing protein, as a positive regulator of nectary formation. An InDel, encoding a five amino acid deletion, together with a nonsynonymous substitution, was predicted to cause 3D structural changes in GaNEC1 protein that could confer the nectariless phenotype. mRNA‐Seq analysis showed that JA‐related genes are up‐regulated and cell wall‐related genes are down‐regulated in the nectary. Silencing of GaNEC1 led to a smaller size of foliar nectary phenotype. Metabolomics analysis identified more than 400 metabolites in nectar, including expected saccharides and amino acids. The identification of GaNEC1 helps establish the network regulating nectary formation and nectar secretion, and has implications for understanding the production of secondary metabolites in nectar. Our results will deepen our understanding of plant–mutualism co‐evolution and interactions, and will enable utilization of a plant defence trait in cotton breeding efforts.