Leaf Nodules Research Articles

Keetia nodulosa sp. nov. (Rubiaceae - Vanguerieae) of West-Central Africa: bacterial leaf nodulation discovered in a fourth genus and tribe of Rubiaceae

Keetia nodulosa Cheek, a cloud forest climber nearly endemic to Cameroon, with a single record from Nigeria, is described and illustrated. It is remarkable as the first known species to be recorded with bacterial leaf nodules (BLN) in the genus Keetia, and also, in the tribe Vanguerieae. Other genera in Rubiaceae with BLN are Psychotria (Psychotrieae-Rubioideae), Sericanthe (Coffeeae) and Pavetta (Pavetteae), both Ixoroideae/Dialypetalanthoideae. The BLN in Keetia (Vanguerieae) are illustrated for the first time here. The characteristics and significance of bacterial leaf nodulation in Keetia nodulosa are discussed in the context of rapidly growing knowledge on the subject in flowering plants. Keetia nodulosa is provisionally assessed using the 2012 IUCN standard as Endangered (EN B2ab(iii)). The importance of its conservation, and options for achieving this are discussed in the context of recent extinctions of other plant species in Cameroon. This discovery of a new cloud forest species is discussed in relation to other cloud forest plant species described in the last twenty years which are also distributed over the highlands of the western half of Cameroon.

Effects of phosphorus and iron on biomass production and nutrients partitioning in soybean cultivars under water stress condition

A pot experiment was conducted to investigate the effects of P and Fe application on the biomass production and nutrients partitioning of two soybeans (Glycine max. L. Merr) cultivars grown in carbonated chernoziom (low in Fe and P) under water stress conditions. P and Fe were applied at two levels (0 and 100 mg P kg-1 soil; 0 and 5 mg Fe kg-1 soil). Control plants were grown at 70% water holding capacity (WHC) while their counterparts were subjected to 35% WHC water stress at initial flowering stage for two weeks. Considerable variability was observed in leaves, roots dry mass accumulation and nodulation among the soybean cultivars (Zodiac, Licurici) at both P and Fe levels in relation to water regimes. The results showed that drought significantly reduced biomass production irrespective of nutrient supply and its adverse effect was more pronounced at low nutrient supply. Leaf development and nodules growth were the most sensitive to water deficit and insufficient nutrient supply. Adequate P and Fe supply increased dry matter production and nutrient concentrations for soybean cultivars. Phosphorus concentration in plant parts was significantly higher at nil Fe compared with Fe application. Phosphorus application decreased Fe allocation to the leaves. The experimental results demonstrated that there was a positive effect of P and Fe adequate nutrition on P use efficiency. Hence, the sufficient phosphorus and iron supply maintained growth at high level, improved P and Fe status and partially alleviated drought effect on soybean plants.

Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-d-glucose analogue (+)-streptol, systemically supplied by mature Ca.Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria–Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis

A majority of Ardisia species harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted hereditarily and have not yet been cultured outside of their host. Because the plants cannot develop beyond the seedling stage without their symbionts, the symbiosis is considered obligatory. We sequenced for the first time the genome of Candidatus Burkholderia crenata (Ca. B. crenata), the leaf nodule symbiont of Ardisia crenata. The genome of Ca. B. crenata is the smallest Burkholderia genome to date. It contains a large amount of insertion sequences and pseudogenes and displays features consistent with reductive genome evolution. The genome does not encode functions commonly associated with plant symbioses such as nitrogen fixation and plant hormone metabolism. However, we identified unique genes with a predicted role in secondary metabolism in the genome of Ca. B. crenata. Specifically, we provide evidence that the bacterial symbionts are responsible for the synthesis of compound FR900359, a cyclic depsipeptide with biomedical properties previously isolated from leaves of A. crenata.

ChemInform Abstract: Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont.

AbstractThe new aminocyclitol, kirkamide (I), is isolated from leaf nodules of Psychotira kirkii.

Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont

AbstractThe new C7N aminocyclitol kirkamide (1) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome‐driven 1H NMR‐guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single‐crystal X‐ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide (1) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd‐mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide (1) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.

Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont

The new C7N aminocyclitol kirkamide (1) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome-driven (1)H NMR-guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single-crystal X-ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide (1) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd-mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide (1) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.

Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species.

• Premise of the study: The pantropical, species-rich Psychotrieae and Palicoureeae are sister tribes of mostly drupe-bearing and nonbacterial leaf-nodulating species with problematic generic limits. This problem is more complicated in Psychotrieae due to the paraphyly of the genus Psychotria, the lack of diagnostic characters for some major lineages, and the poor sampling from some biodiversity hotspots. Schizocarps and bacterial leaf nodules have been used for recognizing formal groups in Psychotrieae, but their evolution and taxonomic value have not been studied using a robust phylogeny of the tribe.• Methods: We analyzed 287 samples from the entire ranges of the tribes, with particular emphasis on the Western Indian Ocean region, with the Bayesian Markov chain Monte Carlo method.• Key results: All allied Psychotria genera investigated are nested within a paraphyletic Psychotria. Schizocarps evolved independently two times within Psychotria, and one reversal back to the drupaceous condition is inferred. The Malagasy leaf-nodulated Psychotrieae (except Apomuria bullata) and the Comorian non-leaf-nodulated Psychotria conocarpa are nested within the (African) leaf-nodulated clade. Within Palicoureeae, Chassalia is paraphyletic with respect to Geophila sensu stricto, and the Malagasy Geophila gerrardii and the African Hymenocoleus are closely related.• Conclusions: A widely circumscribed Psychotria encompassing the entire Psychotrieae is supported. Within Psychotria, two separate origins of schizocarps from drupes, one reversal back to the drupaceous condition, and two independent origins of the Malagasy leaf-nodulated species are inferred. A new genus Puffia is described to accommodate Geophila gerrardii, and a narrow circumscription of Chassalia is adopted. Thirty-two new combinations, two lectotypifications, and 25 new names are presented.

Proteomics Analysis of Psychotria Leaf Nodule Symbiosis: Improved Genome Annotation and Metabolic Predictions

Several plant species of the genus Psychotria (Rubiaceae) harbor Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is considered to be obligatory because plants devoid of symbionts fail to develop into mature individuals. The genome of 'Candidatus Burkholderia kirkii' has been sequenced recently and has revealed evidence of reductive genome evolution, as shown by the proliferation of insertion sequences and the presence of numerous pseudogenes. We employed shotgun proteomics to investigate the expression of 'Ca. B. kirkii' proteins in the leaf nodule. Drawing from this dataset and refined comparative genomics analyses, we designed a new pseudogene prediction algorithm and improved the genome annotation. We also found conclusive evidence that nodule bacteria allocate vast resources to synthesis of secondary metabolites, possibly of the C7N aminocyclitol family. Expression of a putative 2-epi-5-valiolone synthase, a key enzyme of the C7N aminocyclitol synthesis, is high in the nodule population but downregulated in bacteria residing in the shoot apex, suggesting that production of secondary metabolites is particularly important in the leaf nodule.

Odontonema cuspidatum and Psychotria punctata, Two New Hosts of Cucumber mosaic virus in the United States.

Cucumber mosaic virus (CMV) has a reported host range of 750 to 1,200 species (2,3) that includes weeds, wild plants, crops, and ornamentals. Two new CMV hosts were recently identified in Florida. In July 2011, leaves of Odontonema cuspidatum (firespike), a member of the Acanthaceae, with virus-like symptoms were sent to FDACS-DPI. Firespike is an ornamental shrub native to Mexico with evergreen ovate leaves tapering to a pointed tip. Leaf symptoms included severe leaf distortion with some subtle yellowing or mosaic on younger leaves. Pink-red crystals were seen in leaf strips stained with the nucleic acid stain Azure A, indicating a viral infection. In January 2012, leaves of Psychotria punctata (dotted wild coffee), a member of the Rubiaceae, with virus-like symptoms were sent to FDACS-DPI. Dotted wild coffee is a small exotic tropical tree found in south Florida with many tiny leaf nodules inhabited by endosymbiotic bacteria. In addition to the nodules, these leaves had many large dark green ringspots surrounded with a yellow halo. Both samples were positive for CMV when tested with ImmunoStrips and/or by conventional ELISA using CMV antiserum (Agdia, Elkhart, IN). To confirm CMV infection, reverse transcriptase (RT)-PCR on total RNA from a leaf sample of each plant species was used with previously published cucumovirus primers (1). An expected ~940 bp product was amplified from each sample and cloned into pGEM-T (Promega, Madison, WI). Ten clones from each plant species were sequenced in both directions. After removal of primer sequences, the 906 bp products were 96.3% identical with each other and showed 96.8 to 98.9% nucleotide identity with CMV sequences from Hungary, the United States, and Austria (GenBank Accession Nos. AF517802, U20668, and HQ916354, respectively). Identification of CMV infection in these two species expands the known host range and therefore the reservoir of this plant virus. This has implications for the ornamental industry in general and Florida farmers in particular.

The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host

Several plant species of the genus Psychotria (Rubiaceae) harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is also generally described as obligatory because plants devoid of symbionts fail to develop into mature individuals. We sequenced for the first time the genome of the symbiont of Psychotria kirkii in order to shed some light on the nature of their symbiotic relationship. We found that the 4 Mb genome of Candidatus Burkholderia kirkii (B. kirkii) is small for a Burkholderia species and displays features consistent with ongoing genome erosion such as large proportions of pseudogenes and transposable elements. Reductive genome evolution affected a wide array of functional categories that may hinder the ability of the symbiont to be free-living. The genome does not encode functions commonly found in plant symbionts such as nitrogen fixation or plant hormone metabolism. Instead, a collection of genes for secondary metabolites' synthesis is located on the 140 kb plasmid of B. kirkii and suggests that leaf nodule symbiosis benefits the host by providing protection against herbivores or pathogens.

Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)

Burkholderia endophytes were identified within the leaves of non-nodulated members of the genus Psychotria. In contrast to leaf-nodulated Psychotria species, which are known to accommodate their endosymbionts into specialized endosymbiont-housing structures, non-nodulated species lack bacterial leaf nodules and harbor endosymbionts intercellularly between mesophyll cells. Based on molecular data (rps16, trnG, and trnLF), the phylogenetic reconstruction of the host plants revealed a separate origin of leaf-nodulated and non-nodulated Psychotria species. Despite a distinct phylogenetic position of the two host clades, the endophytes of the non-nodulated plants were not placed into a single monophyletic group but were found to be closely related to the leaf-nodulated endosymbionts. The observation of genetically similar endophytes in both nodulated and non-nodulated Psychotria lineages suggests that the host plant is playing a crucial role in the induction of leaf nodule formation. Moreover, the concentration of endosymbionts into specialized leaf nodules may be considered as a more derived evolutionary adaptation of the host plant, serving as an interface structure to facilitate metabolic exchange between plant and endosymbiont.

Identification, origin, and evolution of leaf nodulating symbionts of Sericanthe (Rubiaceae)

Bacterial leaf symbiosis is an intimate association between bacteria and plants in which endosymbionts are housed within leaf nodules. This phenomenon has been reported in three genera of Rubiaceae (Pavetta, Psychotria, and Sericanthe), but the bacterial partner has only been identified in Psychotria and Pavetta. Here we report the identification of symbiotic bacteria in two leaf nodulating Sericanthe species. Using 16S rRNA data and common housekeeping genetic markers (recA and gyrB) we studied the phylogenetic relationships of bacterial endosymbionts in Rubiaceae. Endosymbionts of leaf nodulating Rubiaceae were found to be closely related and were placed as a monophyletic group within the genus Burkholderia (β-Proteobacteria). The phylogenetic analyses revealed a pattern of strict host specificity and placed the two investigated endosymbionts at two distinct positions in the topology of the tree, suggesting at least two different evolutionary origins. The degree of sequence divergence between the Sericanthe endosymbionts and their relatives was large enough to propose the Sericanthe endosymbionts as new species ('Candidatus Burkholderia andongensis' and 'Candidatus Burkholderia petitii'). In a second part of this study, the pylogenetic relationships among nodulating and non-nodulating Sericanthe species were investigated using sequence data from six chloroplast regions (rps16, trnG, trnL-trnF, petD, petA-psbJ, and atpI-atpH). Overall, genetic variation among the plastid markers was insufficient to enable phylogenetic estimation. However, our results could not rule out the possibility that bacterial leaf symbiosis originated once in a common ancestor of the Sericanthe species.

Identification of the bacterial endosymbionts in leaf nodules of Pavetta (Rubiaceae)

Three genera in the Rubiaceae (Pavetta, Psychotria and Sericanthe) harbour bacterial endosymbionts within leaf nodules or galls. The present paper identifies the bacterial endophytes in three leaf-nodulating Pavetta species. In order to reveal their identity and assess their phylogenetic position, 16S rRNA, recA and gyrB genes were sequenced from an extensive sampling of Burkholderia strains. This multigene approach results in a robust phylogeny, which places the bacterial endosymbionts of Pavetta at two distinct positions within the genus Burkholderia (class Betaproteobacteria), suggesting that leaf-nodulating endosymbionts within Pavetta have different origins. The endophytes of nodulated Psychotria species were recognized as the closest relatives to the Pavetta endosymbionts. Our results suggest that the endosymbionts of Pavetta represent novel species, which can be classified as 'Candidatus Burkholderia hispidae', 'Candidatus Burkholderia rigidae' and 'Candidatus Burkholderia schumannianae'.

Coléteres dendróides em Alibertia sessilis (Vell.) K. Schum., uma espécie não-nodulada de Rubiaceae

This paper describes the distribution, structure and histochemistry of the colleters in vegetative and reproductive apices of Alibertia sessilis (Vell.) K. Schum., a native Rubiaceae species of the Brazilian cerrado. In this family, dendroid colleters have been associated with the presence of bacterial nodules in the leaves. Samples were prepared according to common light and scanning electron microscopy techniques, and histochemical assays involved freshly collected material. Colleters are dendroid and occur on the adaxial surface of stipules, bracts and sepals; they consist of a multicellular and multiseriate central axis of parenchymatous nature, covered by digitiform or sharply pointed epidermal cells of irregular sizes, joined together only in the proximal portion and separated from each other in the distal portion. The columnar cells are axially elongated, with thick cell walls, prominent nucleus and reduced cytoplasm, developed vacuole with lipid inclusions and phenolic compounds. The epidermal cells have thin walls, a conspicuous nucleus, abundant cytoplasm and a poorly developed vacuome. The colleters are devoid of cuticle. The secretion is more abundant in the vegetative apex, impregnating the leaf buds and the stipules, forming a spherical cap that is hard, bright, and hydrophobic. In all analyzed samples, the histochemical assays detected polysaccharides, proteins and lipids in the protoplast of both epidermal and columnar cells; phenolic compounds were detected solely in the columnar cells. The resinous nature of the exudate was confirmed with the use of the diethylic ether solvent. Bacterial leaf nodules were not found.

Anatomical and Ultrastuctural Observations in Hypericum perforatum L. Leaves

ABSTRACT The secretory structures for bioactive compounds in Hypericum perforatum L., a herbaceous perennial plant known for anti-inflammatory and healing properties, are distributed throughout the plant leaves and consist of translucent, schizogenous oil cavities and black nodules. Nodules are differentiated during early leaf growth and are initially formed at the edges and tip of young emerging leaves. This study investigated the anatomical, ultrastructural, and cytochemical characteristics of Hypericum perforatum leaf nodules to determine the localization of active compounds.

Structural, Functional and Phylogenetic Aspects of the Colleter

This article surveys the structural, functional and phylogenetic significance of colleters in different dicotyledonous families. Colleters are multicellular secretory structures attached to the stipule, petiole, lamina, bract, bracteole, calyx and corolla. Colleters are grouped into standard (S), dendroid (D) and brush-like (B) types on the basis of their morphology and structure. D and B-type colleters occur in certain members of Rubiaceae that also have bacterial leaf nodules. Besides the normal structure, epithelial hairs, thin-walled subepidermal cells, laticifers and vasculature are present in many colleters of Apocynaceae. It is probable that the colleter functions to protect the developing meristem by secreting a viscous fluid. Exudate of D-type colleters are mucilaginous, providing the substrate necessary for the nutrition of endophytic bacteria.

Location and distribution of symbiotic bacteria during floral development in Ardisia crispa

Abstract. The location and distribution of symbiotic bacteria during floral development in Ardisia crispa (Thunb.) A.DC., a species characterized by bacterial leaf nodules, has been studied using light, scanning and transmission electron microscopy. During early floral development, bacteria in mucilage derived from host plant trichomes, become enclosed in a small conical chamber on top of the placenta, as a result of the closure and fusion of the carpel initials. The placental epidermal cells, which appear to be secretory in nature, become detached apically in places forming a network of grooves which traverse the placental surface. The symbiotic bacteria are preferentially located in these grooves. As growth and development of the placenta proceed, the grooves widen and deepen to form channels. The cells lining these channels secrete a mucilaginous material. The network of channels covers the entire placental surface and terminates at the placental margins surrounding the ovules. Bacteria are found within the channels, at the ends of the channels near the margin of the placenta, on the surface of the ovules and in the micropyle. It is suggested that these mucilage‐filled channels are responsible for, and a prerequisite of, ensuring that the bacterial partner is efficiently transmitted from one host generation to the next by providing a mechanism by which the bacteria arc accurately placed within the developing seed.

The occurrence of calyx nodules inPsychotria spp. (Rubiaceae)

The occurrence and structure of calyx nodules in the flowers of two leaf nodulated rubiaceous speciesPsychotria punctata Vatke andPsychotria kirkii Hiern. has been described for the first time at the ultrastructural level. Bacteria, resident in colleter-secreted mucilage in the space between calyx and corolla, invade stomatal pores which develop on the calyx protoderm. The bacteria proliferate in the substomatal cavity and then invade the calyx mesophyll. This invasion is most pronounced inP. punctata where the bacteria even penetrate and enter the cells of the vascular tissue. Although no sheath forms around the calyx nodules, the calyx mesophyll cells surrounded by the bacteria become identical in shape, size and secretory function to the invasive mesophyll cells of leaf nodules. The functional and evolutionary significance of calyx nodulation is discussed.