Histochemical, metabolic and ultrastructural changes in leaf patelliform nectaries explain extrafloral nectar synthesis and secretion in Clerodendrum chinense.

BACKGROUND AND AIMS: Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes. METHODS: We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined. KEY RESULTS: Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region. CONCLUSIONS: Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion.

GoNe encoding a class VIIIb AP2/ERF is required for both extrafloral and floral nectary development in Gossypium.

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne1 Ne2 lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.

Apoplasmic barrier in the extrafloral nectary of Citharexylum myrianthum (Verbenaceae).

MAIN CONCLUSION: The cytological changes underlying the formation of an apoplasmic barrier in the multi-layered extrafloral nectaries of Citharexylum myrianthum are compatible with the synthesis, transport and deposition of suberin. In terms of ontogenesis and function, the intermediate layers of these nectaries are homologous with the stalks of nectar-secreting trichomes. Anticlinal cell wall impregnations are common in trichomatic nectaries and their functions as endodermis-like barriers have been discussed because of possible direct effects on the nectary physiology, mainly in the nectar secretion and resorption. However, the cytological events linked to nectary wall impregnations remain little explored. This study documents the ontogenesis and the fine structure of the EFN cells, and cytological events linked to the wall impregnations of multi-layered extrafloral nectaries (EFNs) in Citharexylum myrianthum Cham. (Verbenaceae). EFNs are patelliform, and differentiated into (a) a multicellular foot, which is compound in structure and vascularised with phloem strands, (b) a bi-layered intermediate region with thickened cell walls and (c) a single-layered secretory region with palisade-like cells. EFNs are protodermal in origin, starting with a single protodermal cell and ending with the complex, multi-layered structure. The cell wall impregnations first appear in the very young EFN and increase towards maturity. Lipid patches (assumed to be suberin) are deposited on the inner faces of the primary walls, first along the anticlinal walls and then extend to the periclinal walls. On both walls, plasmodesmata remain apparently intact during the maturation of the EFNs. In the peripheral cytoplasm there are abundant polymorphic plastids, well-developed Golgi bodies often close to rough endoplasmic reticulum profiles, mitochondria and polyribosomes. Cytological events linked to the wall impregnations are consistent with suberin synthesis, transport and deposition. Our findings offer new insights into the structure-properties of specialised nectary cell walls and so should contribute to our knowledge of the physiological and protective roles of this structure in nectar glands.

Spatiotemporal niche-based mechanisms support a stable coexistence of ants and spiders in an extrafloral nectary-bearing plant community.

Mechanisms promoting stable coexistence allow multiple species to persist in the same trophic level of a given network of species interactions. One of the most common stabilizing mechanisms of coexistence is niche differentiation, such as temporal and spatial patchiness. To understand the limits of coexistence between species we have to understand the limits of competitive interactions which translate in species exclusion or patterns of non-co-occurrence. We evaluated spatiotemporal niche-based mechanisms that could promote stable coexistence between ants and spiders which forage on extrafloral nectary (EFN)-bearing plants. We observed co-occurrence and overlapping patterns between ants and spiders in a temporal and spatial scale in nine different EFN-bearing plant species in a Neotropical savanna, using both community and species-level approach. Ants and spiders showed asynchrony of their abundances over the year with low temporal overlapping patterns between them (temporal niche specialization). Greater abundance of ants occurred between September and March, whereas greater abundance of spiders occurred between March and August, exactly at the time when the abundance of ants decreases on plants. However, there might also be some levels of temporal overlapping, but then individual ants and spiders occupy different branches (spatial segregation). Finally, we also observed a spatial negative effect of the abundance of ants on the presence of spiders. Our results suggest that spatiotemporal partitioning between ants and spiders may be one of the potential mechanisms behind a stable coexistence between these two groups of organisms that forage on EFN-bearing plants in the Brazilian savanna.

Unravelling the Homology between Calycine Glands in Malpighiales: New Data from Basal Malpighiaceae.

Discussing homology relationships among secretory structures remains a relatively underexplored area in botanical research. These structures are widely dispersed within Malpighiales, one of the largest orders of eudicots. Within Malpighiales, both extranuptial and nuptial nectaries are present, and they do not seem homoplastic or share evolutionary connections. Particularly in Malpighiaceae, extensive research has focused on the ecological interactions mediated by glands. Botanists largely agree that elaiophores in sepals of Neotropical Malpighiaceae have evolved from extrafloral nectaries on leaves. However, the evolutionary origin of elaiophores has yet to be thoroughly examined, particularly in comparison to outgroups. This study provides empirical evidence on the ontogeny of elaiophores and investigates their evolutionary origins and homology relationships across different lineages of Malpighiales using comparative anatomy. Our findings suggest that elaiophores are likely homologous to extranuptial nectaries found in sepals of other Malpighiales lineages, originating from nectaries on leaves. This discussion is a starting point for future studies exploring the evolution of nectaries found in flowers, whether extranuptial or nuptial, and their potential origins from nectaries in vegetative organs such as leaves. Understanding these relationships could shed light on the selective pressures influencing floral morphologies.

Ants may buffer the Janzen-Connell effect in a tropical forest in Southwest China.

Mutualistic symbioses between ants and plants are widespread in nature. Ants can deter unwanted pests and provide protection for plants in return for food or housing rewards. Using a long-term demographic dataset in a tropical seasonal rain forest in Southwest China, we found that associations with ants positively influenced seedling survival and adult growth, and also, species with extrafloral nectaries experienced weaker conspecific negative density dependence compared with species without extrafloral nectaries. Furthermore, we found strong evidence suggesting that species in our forest experienced conspecific density dependence, which we interpreted as heavy pest pressure that may drive the development of anti-pest symbioses such as the plant-ant relationship. Our findings suggest that ants and conspecific neighbors play important but inverse roles on plant survival and growth and that ants can buffer tree neighborhood interactions in this tropical forest.

The phylogenetic distribution of extrafloral nectaries in plants.

BACKGROUND AND AIMS: Understanding the evolutionary patterns of ecologically relevant traits is a central goal in plant biology. However, for most important traits, we lack the comprehensive understanding of their taxonomic distribution needed to evaluate their evolutionary mode and tempo across the tree of life. Here we evaluate the broad phylogenetic patterns of a common plant-defence trait found across vascular plants: extrafloral nectaries (EFNs), plant glands that secrete nectar and are located outside the flower. EFNs typically defend plants indirectly by attracting invertebrate predators who reduce herbivory. METHODS: Records of EFNs published over the last 135 years were compiled. After accounting for changes in taxonomy, phylogenetic comparative methods were used to evaluate patterns of EFN evolution, using a phylogeny of over 55 000 species of vascular plants. Using comparisons of parametric and non-parametric models, the true number of species with EFNs likely to exist beyond the current list was estimated. KEY RESULTS: To date, EFNs have been reported in 3941 species representing 745 genera in 108 families, about 1-2 % of vascular plant species and approx. 21 % of families. They are found in 33 of 65 angiosperm orders. Foliar nectaries are known in four of 36 fern families. Extrafloral nectaries are unknown in early angiosperms, magnoliids and gymnosperms. They occur throughout monocotyledons, yet most EFNs are found within eudicots, with the bulk of species with EFNs being rosids. Phylogenetic analyses strongly support the repeated gain and loss of EFNs across plant clades, especially in more derived dicot families, and suggest that EFNs are found in a minimum of 457 independent lineages. However, model selection methods estimate that the number of unreported cases of EFNs may be as high as the number of species already reported. CONCLUSIONS: EFNs are widespread and evolutionarily labile traits that have repeatedly evolved a remarkable number of times in vascular plants. Our current understanding of the phylogenetic patterns of EFNs makes them powerful candidates for future work exploring the drivers of their evolutionary origins, shifts, and losses.

Diversity of Leaf Glands and Their Putative Functions in Rhamnaceae Species.

Leaf glands are found in many Rhamnaceae species, the buckthorn family, and are frequently used in taxonomic studies of the group, especially because they are easily visible to the naked eye. Despite the many records and extensive use in the taxonomy of the family, few studies deal with the classification of these glands and their roles for the plant. Thus, this study aimed to unravel the type, functioning, and putative functions of the leaf glands of three Brazilian forest species: Colubrina glandulosa Perkins, Gouania polygama (Jacq.) Urb., and Rhamnidium elaeocarpum Reissek. Leaves were collected and processed for surface, anatomical, histochemical, and ultrastructural analyses. In addition, the presence of visitor animals was registered in the field. The leaf glands of C. glandulosa and G. polygama are defined as extrafloral structured nectaries due to their anatomical structure, interaction with ants, and the presence of reduced sugars and of a set of organelles in the secretory cells. The unusual mechanism of nectar release and exposure in an apical pore stands out in G. polygama. The glands of R. elaeocarpum are ducts or cavities that secrete phenolic oil resin. Their presence is an atypical condition in the family, although they are often confused with mucilage reservoirs, much more common in Rhamnaceae. The extrafloral nectary, secretory cavity, and duct are associated with plant protection against phytophages, either by attracting patrol ants or by making the organs deterrent. Our data, combined with other previously obtained data, attest to the great diversity of gland types found in Rhamnaceae species.

Extrafloral nectar as entrée and elaiosomes as main course for ant visitors to a fireprone, mediterranean-climate shrub.

Thousands of plants produce both extrafloral nectaries (EFNs) on their leaves and nutrient-rich appendages on their diaspores (elaiosomes). Although their individual ecology is well-known, any possible functional link between these structures has almost always been ignored. Here, we recognized their co-presence in the shrub, Adenanthos cygnorum (Proteaceae), and studied their function and interaction. We observed that the same ants frequently visit both structures, seeds are attractive to vertebrate granivores but are released into a leafy cup from where they are harvested by ants and taken to their nests, from which seeds, lacking elaiosomes, germinate after fire. We showed that juvenile plants do not produce EFNs and are not visited by ants. We conclude that EFNs are not just an indirect adaptation to minimize herbivory via aggressive ant visitors (the role of a minority) but specifically enhance reproductive success in two ways: First, by inducing ants to visit the plant as a reliable food source throughout the year. Second, by promoting discovery of the seasonally available, elaiosome-bearing seeds for transport to their nests (the majority of visitors), so avoiding the risk of granivory should seeds instead fall to the ground. Parasitoid wasps play a supporting role in controlling the main insect herbivore whose larvae devour the reproductive apices. Thus, the EFN-elaiosome relationship has three components that enhance species fitness: foliage protection, seed transport, and granivore escape. A similar system has been described only once before (in an unrelated biome) and, consistent with the objectives of ecology as an integrative science, deserves wider study.

Multitrophic relationships, conditional mutualisms, and the study of interaction biodiversity in tropical savannas / Relações multitróficas, mutualismos condicionais e o estudo da interação da biodiversidade em savanas tropicais

Biodiversity as a focus for conservation efforts has received increased attention since the second half of the last century. Until recently a more landscape-level view of biodiversity has prevailed. This essay proposes to discuss natural diversity in ways that embrace the extreme richness inherent in plant animal interactions, including not only trophic relationships, but also aspects of life history, biology, and behavior of related species. The ant-plant-herbivore systems in Cerrado vegetation are pointed out as good model for gaining a better understanding of "interaction biodiversity".

A biodiversidade como um foco em esforços de conservação tem recebido cada vez mais atenção desde a segunda metade do século passado. Até recentemente a visão da biodiversidade ao nível da paisagem (comunidades) tem prevalecido. Este ensaio se propõe a discutir a diversidade natural também através da riqueza extrema inerente às interações entre animais e plantas, incluindo não apenas relações tróficas, mas também aspectos de histórias de vida, biologia e comportamento das espécies relacionadas. Sistemas plantas-formigas-herbívoros na vegetação de Cerrado são aqui apontados como modelos apropriados para aumentar a compreensão sobre a "biodiversidade interativa".