Water Affects Morphogenesis of Growing Aquatic Plant Leaves.

Lotus leaves floating on water usually experience short-wavelength edge wrinkling that decays toward the center, while the leaves growing above water normally morph into a global bending cone shape with long rippled waves near the edge. Observations suggest that the underlying water (liquid substrate) significantly affects the morphogenesis of leaves. To understand the biophysical mechanism under such phenomena, we develop mathematical models that can effectively account for inhomogeneous differential growth of floating and freestanding leaves to quantitatively predict formation and evolution of their morphology. We find, both theoretically and experimentally, that the short-wavelength buckled configuration is energetically favorable for growing membranes lying on liquid, while the global buckling shape is more preferable for suspended ones. Other influencing factors such as the stem or vein, heterogeneity, and dimension are also investigated. Our results provide a fundamental insight into a variety of plant morphogenesis affected by water foundation and suggest that such surface instabilities can be harnessed for morphology control of biomimetic deployable structures using substrate or edge actuation.

Dissecting the 'bacon and eggs' phenotype: transcriptomics of post-anthesis colour change in Lotus.

Background and Aims: Post-anthesis colour change (PACC) is widely thought to be an adaptation to signal floral suitability to pollinators. Lotus filicaulis and Lotus sessilifolius are insect-pollinated herbaceous legumes with flowers that open yellow, shift to orange and finally red. This study examines the molecular basis for floral colour change in these Lotus species. Methods: Lotus filicaulis was cultivated in a glasshouse from which pollinating insects (bees) were excluded, and the rate of colour change was recorded in both unpollinated and manually pollinated flowers. Unpollinated flowers from both the yellow stage and the red stage were sampled for sequencing. The transcriptomes of L. filicaulis and L. sessilifolius of both colour stages were analysed for differentially expressed genes and enriched ontologies. Key Results: The rate of progression through PACC doubled when L. filicaulis was hand-pollinated. De novo assembly of RNA-Seq reads from non-model Lotus species outperformed heterologous alignment of reads to the L. japonicus genome. Differential expression analysis suggested that the carotenoid biosynthetic pathway is upregulated at anthesis while the flavonoid biosynthetic pathway is upregulated with the onset of PACC in L. filicaulis and L. sessilifolius . Conclusion: Pollination significantly accelerates PACC in L. filicaulis , consistent with the hypothesis that PACC increases pollination efficiency by directing pollinators to unpollinated flowers. RNA-Seq results show the synchronized upregulation of the entire cyanidin biosynthesis pathway in the red stage of PACC in L. filicaulis and L. sessilifolius . The genes implicated offer the basis for further investigations into how gene families, transcription factors and related pathways are likely to be involved in PACC.

Hydrogels with Lotus Leaf Topography: Investigating Surface Properties and Cell Adhesion.

The interactions of cells with the surface of materials is known to be influenced by a range of factors that include chemistry and roughness; however, it is often difficult to probe these factors individually without also changing the others. Here we investigate the role of roughness on cell adhesion while maintaining the same underlying chemistry. This was achieved by using a polymerization in mold technique to prepare poly(hydroxymethyl methacrylate) hydrogels with either a flat topography or a topography that replicated the microscale features of lotus leaves. These materials were then assessed for cell adhesion, and atomic force microscopy and contact angle analysis were then used to probe the physical reasons for the differing behavior in relation to cell adhesion.

Fractal Surfaces of Molecular Crystals Mimicking Lotus Leaf with Phototunable Double Roughness Structures.

Double roughness structure, the origin of the lotus effect of natural lotus leaf, was successfully reproduced on a diarylethene microcrystalline surface. Static superwater-repellency and dynamic water-drop-bouncing were observed on the surface, in the manner of natural lotus leaves. Double roughness structure was essential for water-drop-bouncing. This ability was not observed on a single roughness microcrystalline surface showing the lotus effect of the same diarylethene derivative. The double roughness structure was reversibly controlled by alternating irradiation with UV and visible light.

Superhydrophobic surfaces developed by mimicking hierarchical surface morphology of lotus leaf.

The lotus plant is recognized as a 'King plant' among all the natural water repellent plants due to its excellent non-wettability. The superhydrophobic surfaces exhibiting the famous 'Lotus Effect', along with extremely high water contact angle (>150°) and low sliding angle (<10°), have been broadly investigated and extensively applied on variety of substrates for potential self-cleaning and anti-corrosive applications. Since 1997, especially after the exploration of the surface micro/nanostructure and chemical composition of the lotus leaves by the two German botanists Barthlott and Neinhuis, many kinds of superhydrophobic surfaces mimicking the lotus leaf-like structure have been widely reported in the literature. This review article briefly describes the different wetting properties of the natural superhydrophobic lotus leaves and also provides a comprehensive state-of-the-art discussion on the extensive research carried out in the field of artificial superhydrophobic surfaces which are developed by mimicking the lotus leaf-like dual scale micro/nanostructure. This review article could be beneficial for both novice researchers in this area as well as the scientists who are currently working on non-wettable, superhydrophobic surfaces.

Akaline, saline and mixed saline-alkaline stresses induce physiological and morpho-anatomical changes in Lotus tenuis shoots.

Saline, alkaline and mixed saline-alkaline conditions frequently co-occur in soil. In this work, we compared these plant stress sources on the legume Lotus tenuis, regarding their effects on shoot growth and leaf and stem anatomy. In addition, we aimed to gain insight on the plant physiological status of stressed plants. We performed pot experiments with four treatments: control without salt (pH = 5.8; EC = 1.2 dS·m(-1)) and three stress conditions, saline (100 mM NaCl, pH = 5.8; EC = 11.0 dS·m(-1)), alkaline (10 mM NaHCO3, pH = 8.0, EC = 1.9 dS·m(-1)) and mixed salt-alkaline (10 mM NaHCO3 + 100 mM NaCl, pH = 8.0, EC = 11.0 dS·m(-1)). Neutral and alkaline salts produced a similar level of growth inhibition on L. tenuis shoots, whereas their mixture exacerbated their detrimental effects. Our results showed that none of the analysed morpho-anatomical parameters categorically differentiated one stress from the other. However, NaCl- and NaHCO3 -derived stress could be discriminated to different extents and/or directions of changes in some of the anatomical traits. For example, alkalinity led to increased stomatal opening, unlike NaCl-treated plants, where a reduction in stomatal aperture was observed. Similarly, plants from the mixed saline-alkaline treatment characteristically lacked palisade mesophyll in their leaves. The stem cross-section and vessel areas, as well as the number of vascular bundles in the sectioned stem were reduced in all treatments. A rise in the number of vessel elements in the xylem was recorded in NaCl-treated plants, but not in those treated exclusively with NaHCO3.

Preparation of lotus-leaf-like structured blood compatible poly(ε-caprolactone)-block-poly(L-lactic acid) copolymer film surfaces.

Lotus-leaf-like structured poly(ε-caprolactone)-block-poly(L-lactic acid) copolymer (PCL-b-PLLA) films cast using the solvent-nonsolvent casting method. PCL-b-PLLA was synthesized by the well-known copolymerization process, and was confirmed by (1)H NMR analysis. The molecular weight of the synthesized PCL-b-PLLA was measured by gel permeation chromatography (GPC). The number-average (Mn), weight-average (Mw) molecular weights and polydispersity (Mw/Mn) were 3.9×10(4), 5.1×10(4), and 1.3, respectively. PCL-b-PLLA films were cast in vacuum conditions with various nonsolvent ratios. Tetrahydrofuran (THF) was used as solvent and ethanol was used as nonsolvent. Surface hydrophobicity was confirmed by the water contact angle. The water contact angle was increased from 90.9°±4.2° to 130.2°±3.6°. Water contact angle was found to be influenced by surface topography. The prepared film surfaces were characterized by scanning electron microscopy (SEM). Changes in crystalline property were characterized by X-ray diffraction (XRD). Platelet adhesion tests of the modified PCL-b-PLLA film surfaces were evaluated by platelet-rich plasma (PRP) and whole blood. Cell adhesive behavior on the modified film surfaces was evaluated by fibroblast cell culture. The prepared lotus-leaf-like structured film surfaces exhibited reduced platelet adhesion and an increased fibroblast cell proliferation ratio.

Lotus-leaf-like structured heparin-conjugated poly(L-lactide-co-epsilon-caprolactone) as a blood compatible material.

A heparin-conjugated biodegradable polymer was synthesized by direct coupling of heparin to poly(L-lactide-co-ɛ-caprolactone) (PLCL) and was manufactured into lotus-leaf-like structured films. We evaluated whether lotus-leaf-like structured heparin-conjugated PLCL (LH-PLCL) could be applied to blood vessel tissue engineering. Differences in the surface structures of the films with respect to hydrophobicity and the lotus effect as well as the antithrombotic efficiency in human whole blood were examined using scanning electron microscopy (SEM) and a contact angle meter. Recovery testing was conducted using a tensile strength testing machine, and quantitative analysis of conjugated heparin was performed using the toluidine blue colorimetric method. The concentration of conjugated heparin was 0.14 µg/mg H-PLCL, and the contact angle with the lotus-leaf-like surface was approximately 120°. Furthermore, the LH-PLCL film yielded a lower platelet adhesion rate (around less than 1.4%) in whole blood than that yielded by an untreated PLCL film. These results indicate a unique property of bound heparin and the lotus-leaf-like structure. This novel LH-PLCL polymer could be applied as a blood/tissue compatible biodegradable material for implantable medical devices and tissue engineering.

Seed shape in model legumes: approximation by a cardioid reveals differences in ethylene insensitive mutants of Lotus japonicus and Medicago truncatula.

Seed shape in the model legumes Lotus japonicus and Medicago truncatula is described. Based in previous work with Arabidopsis, the outline of the longitudinal sections of seeds is compared with a cardioid curve. L. japonicus seeds adjust well to an unmodified cardioid, whereas accurate adjustment in M. truncatula is obtained by the simple transformation of scaling the vertical axis by a factor equal to the Golden Ratio. Adjustments of seed shape measurements with simple geometrical forms are essential tools for the statistical analysis of variations in seed shape under different conditions or in mutants. The efficiency of the adjustment to a cardioid in the model plants suggests that seed morphology may be related to genome complexity. Seeds of ethylene insensitive mutants present differences in size and shape as well as altered responses to imbibition. The biological implication and meaning of these relationships are discussed.

Bioinspired self-repairing slippery surfaces with pressure-stable omniphobicity.

Creating a robust synthetic surface that repels various liquids would have broad technological implications for areas ranging from biomedical devices and fuel transport to architecture but has proved extremely challenging. Inspirations from natural nonwetting structures, particularly the leaves of the lotus, have led to the development of liquid-repellent microtextured surfaces that rely on the formation of a stable air-liquid interface. Despite over a decade of intense research, these surfaces are, however, still plagued with problems that restrict their practical applications: limited oleophobicity with high contact angle hysteresis, failure under pressure and upon physical damage, inability to self-heal and high production cost. To address these challenges, here we report a strategy to create self-healing, slippery liquid-infused porous surface(s) (SLIPS) with exceptional liquid- and ice-repellency, pressure stability and enhanced optical transparency. Our approach-inspired by Nepenthes pitcher plants-is conceptually different from the lotus effect, because we use nano/microstructured substrates to lock in place the infused lubricating fluid. We define the requirements for which the lubricant forms a stable, defect-free and inert 'slippery' interface. This surface outperforms its natural counterparts and state-of-the-art synthetic liquid-repellent surfaces in its capability to repel various simple and complex liquids (water, hydrocarbons, crude oil and blood), maintain low contact angle hysteresis (<2.5°), quickly restore liquid-repellency after physical damage (within 0.1-1 s), resist ice adhesion, and function at high pressures (up to about 680 atm). We show that these properties are insensitive to the precise geometry of the underlying substrate, making our approach applicable to various inexpensive, low-surface-energy structured materials (such as porous Teflon membrane). We envision that these slippery surfaces will be useful in fluid handling and transportation, optical sensing, medicine, and as self-cleaning and anti-fouling materials operating in extreme environments.

NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus.

In Lotus japonicus, seven genetic loci have been identified thus far as components of a common symbiosis (Sym) pathway shared by rhizobia and arbuscular mycorrhizal fungi. We characterized the nup85 mutants (nup85-1, -2, and -3) required for both symbioses and cloned the corresponding gene. When inoculated with Glomus intraradices, the hyphae managed to enter between epidermal cells, but they were unable to penetrate the cortical cell layer. The nup85-2 mutation conferred a weak and temperature-sensitive symbiotic phenotype, which resulted in low arbuscule formation at 22 degrees C but allowed significantly higher arbuscule formation in plant cortical cells at 18 degrees C. On the other hand, the nup85 mutants either did not form nodules or formed few nodules. When treated with Nod factor of Mesorhizobium loti, nup85 roots showed a high degree of root hair branching but failed to induce calcium spiking. In seedlings grown under uninoculated conditions supplied with nitrate, nup85 did not arrest plant growth but significantly reduced seed production. NUP85 encodes a putative nucleoporin with extensive similarity to vertebrate NUP85. Together with symbiotic nucleoporin NUP133, L. japonicus NUP85 might be part of a specific nuclear pore subcomplex that is crucial for fungal and rhizobial colonization and seed production.

Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth.

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.

Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype.

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.

Control of petal shape and floral zygomorphy in Lotus japonicus.

Zygomorphic flowers, with bilateral (dorsoventral) symmetry, are considered to have evolved several times independently in flowering plants. In Antirrhinum majus, floral dorsoventral symmetry depends on the activity of two TCP-box genes, CYCLOIDEA (CYC) and DICHOTOMA (DICH). To examine whether the same molecular mechanism of floral asymmetry operates in the distantly related Rosid clade of eudicots, in which asymmetric flowers are thought to have evolved independently, we investigated the function of a CYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus. We showed a role for LjCYC2 in establishing dorsal identity by altering its expression in transgenic plants and analyzing its mutant allele squared standard 1 (squ1). Furthermore, we identified a lateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays a key role in the control of lateral petal identity, and found LjCYC2 interacted with Kew1, resulting in a double mutant that bore all petals with ventralized identity to some extents. Thus, we demonstrate that CYC homologues have been independently recruited as determinants of petal identities along the dorsoventral axis in two distant lineages of flowering plants, suggesting a common molecular origin for the mechanisms controlling floral zygomorphy.

Functional diversity of plant-pollinator interaction webs enhances the persistence of plant communities.

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant-pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.

cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus.

A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.