Porous Collagen Sponge Loaded with Large Efficacy-Potentiated Exosome-Mimicking Nanovesicles for Diabetic Skin Wound Healing.

Diabetic skin wounds are difficult to heal quickly due to insufficient angiogenesis and prolonged inflammation, which is an urgent clinical problem. To address this clinical problem, it becomes imperative to develop a dressing that can promote revascularization and reduce inflammation during diabetic skin healing. Herein, a multifunctional collagen dressing (CTM) was constructed by loading large efficacy-potentiated exosome-mimicking nanovesicles (L-Meseomes) into a porous collagen sponge with transglutaminase (TGase). L-Meseomes were constructed in previous research with the function of promoting cell proliferation, migration, and angiogenesis and inhibiting inflammation. CTM has a three-dimensional porous network structure with good biocompatibility, swelling properties, and degradability and could release L-Meseome slowly. In vitro experiments showed that CTM could promote the proliferation of fibroblasts and the polarization of macrophages to the anti-inflammatory phenotype. For in vivo experiments, on the 21st day after surgery, the wound healing rates of the control and CTM were 83.026 ± 4.17% and 93.12 ± 2.16%, respectively; the epidermal maturation and dermal differentiation scores in CTM were approximately four times that of the control group, and the skin epidermal thickness of the CTM group was approximately 20 µm, which was closest to that of normal rats. CTM could significantly improve wound healing in diabetic rats by promoting anti-inflammation, angiogenesis, epidermal recovery, and dermal collagen deposition. In summary, the multifunctional collagen dressing CTM could significantly promote the healing of diabetic skin wounds, which provides a new strategy for diabetic wound healing in the clinic.

Physiological and metabolomic analyses reveal that Fe3O4 nanoparticles ameliorate cadmium and arsenic toxicity in Panax notoginseng.

Heavy metal(loid)-contaminated available arable land seriously affects crop development and growth. Engineered nanomaterials have great potential in mitigating toxic metal(loid) stress in plants. However, there are few details of nanoparticles (NPs) involved in Panax notoginseng response to cadmium (Cd) and arsenic (As). Herein, integrating physiological and metabolomic analyses, we investigated the effects of Fe3O4 NPs on plant growth and Cd/As responses in P. notoginseng. Cd/As treatment caused severe growth inhibition. However, foliar application of Fe3O4 NPs increased beneficial elements in the roots and/or leaves, decreased Cd/As content by 10.38% and 20.41% in the roots, reduced membrane damage and regulated antioxidant enzyme activity, thereby alleviating Cd/As-induced growth inhibition, as indicated by increased shoot fresh weight (FW), the rootlet length and root FW by 40.14%, 15.74%, and 46.70% under Cd stress and promoted the shoot FW by 27.00% under As toxicity. Metabolomic analysis showed that 227 and 295 differentially accumulated metabolites (DAMs) were identified, and their accumulation patterns were classified into 8 and 6 clusters in the roots and leaves, respectively. Fe3O4 NPs altered metabolites significantly involved in key pathways, including amino sugar and nucleotide sugar metabolism, flavonoid biosynthesis and phenylalanine metabolism, thus mediating the trade-off between plant growth and defense under stress. Interestingly, Fe3O4 NPs recovered more Cd/As-induced DAMs to normal levels, further supporting that Fe3O4 NPs positively affected seedling growth under metal(loid)s stress. In addition, Fe3O4 NPs altered terpenoids when the seedlings were subjected to Cd/As stress, thus affecting their potential medicinal value. This study provides insights into using nanoparticles to improve potential active ingredients of medicinal plants in metal(loid)-contaminated areas.

Multifunctional hydrogel/platelet-rich fibrin/nanofibers scaffolds with cell barrier and osteogenesis for guided tissue regeneration/guided bone regeneration applications.

Periodontal defect seriously affects people's life health and quality. Guided tissue regeneration (GTR) and guided bone regeneration (GBR) have made great progress in periodontal disease treatment, but some deficiencies existed in commercial materials of GTR and GBR. For obtaining better therapeutic effects, multifunctional composite scaffolds containing different biological macromolecules were developed in this study. Chitosan/poly (γ-glutamic acid)/nano-hydroxyapatite hydrogels (CP/nHA) made by electrostatic interactions and lyophilization were filled in the bone defects to achieve osteogenesis. Platelet-rich fibrin (PRF) extracted from blood could accelerate bone formation by releasing various bioactive substances as middle layer of composite scaffolds. Polycaprolactone/gelatin nanofibers (PG) prepared by electrospinning were attached to the junction of soft and hard tissue, which could prevent fibrous tissue from infiltrating into bone defects. The composite scaffolds showed good morphology, biocompatibility, cell barriers and osteogenic differentiation in vitro. The excellent ability of bone formation was verified by implantation of triple-layered composite scaffolds into alveolar bone defects in rabbit in vivo. The hierarchical structure was conducive to personalized customization to meet the needs of different defects. All in all, the multifunctional scaffolds could play important roles of GTR and GBR in alveolar bone regeneration and provide good application prospect for bone repair in clinic.

Hyaluronic acid-based dual network hydrogel with sustained release of platelet-rich plasma as a diabetic wound dressing.

In recent years, the incidence of diabetic skin ulcers has increased. Because of its extremely high disability and fatality rate, it brings a huge burden to patients and society. Platelet-rich plasma (PRP) contains a large number of biologically active substances and is of great clinical value in the treatment of various wounds. However, its weak mechanical properties and the consequent abrupt release of active substances greatly limit its clinical application and therapeutic efficacy. Here, we chose hyaluronic acid (HA) and ε-polylysine (ε-PLL) to prepare a hydrogel with the ability to prevent wound infection and promote tissue regeneration. At the same time, using the macropore barrier effect of the lyophilized hydrogel scaffold, platelets in PRP are activated with calcium gluconate in the macropores of the scaffold carrier, and fibrinogen from PRP is converted in a fibrin-packed network forming a gel that interpenetrates the hydrogel scaffold carrier, thus creating a double network hydrogel with slow-release of growth factors from degranulated platelets. The hydrogel not only showed better performance in functional assays in vitro, but also showed more superior therapeutic effects in reducing inflammatory response, promoting collagen deposition, facilitating re-epithelialization and angiogenesis in the treatment of full skin defects in diabetic rats.

Microbiota-mediated nitrogen fixation and microhabitat homeostasis in aerial root-mucilage.

BACKGROUND: Plants sustain intimate relationships with diverse microbes. It is well-recognized that these plant-associated microbiota shape individual performance and fitness of host plants, but much remains to be explored regarding how they exert their function and maintain their homeostasis. RESULTS: Here, using pink lady (Heterotis rotundifolia) as a study plant, we investigated the phenomenon of microbiota-mediated nitrogen fixation and elucidated how this process is steadily maintained in the root mucilage microhabitat. Metabolite and microbiota profiling showed that the aerial root mucilage is enriched in carbohydrates and diazotrophic bacteria. Nitrogen isotope-labeling experiments, 15N natural abundance, and gene expression analysis indicated that the aerial root-mucilage microbiota could fix atmospheric nitrogen to support plant growth. While the aerial root mucilage is a hotspot of nutrients, we did not observe high abundance of other environmental and pathogenic microbes inside. We further identified a fungus isolate in mucilage that has shown broad-spectrum antimicrobial activities, but solely allows the growth of diazotrophic bacteria. This "friendly" fungus may be the key driver to maintain nitrogen fixation function in the mucilage microhabitat. Video Abstract CONCLUSION: The discovery of new biological function and mucilage-habitat friendly fungi provides insights into microbial homeostasis maintenance of microenvironmental function and rhizosphere ecology.

Sustained Release of BMSC-EVs from 3D Printing Gel/HA/nHAP Scaffolds for Promoting Bone Regeneration in Diabetic Rats.

Extracellular vesicles (EVs) play an important role in intercellular communication, and the function of EVs mainly depends on the state of source cells. To determine the effect of diabetic microenvironment on EVs secreted by bone marrow mesenchymal stem cells (BMSCs), this work explores the effect of normal glucose (5.5 mm) cultured BMSCs derived EVs (NG-EVs) and high glucose (30 mm) cultured BMSCs derived EVs (HG-EVs) in regulating the migration, proliferation and osteoblastic differentiation of BMSCs in vitro. In order to improve the bioavailability of EVs, this work constructs a sustained release system of polydopamine (PDA) functionalized 3D printing gelatin/hyaluronic acid/nano-hydroxyapatite (Gel/HA/nHAP) scaffolds (S/PDA) and verifies its function in the calvarial defect model of diabetic rats. This work confirms that both NG-EVs and HG-EVs can promote proliferation and migration, inhibit apoptosis and promote osteogenic differentiation, but the function of HG-EVs is weaker than that of NG-EVs. Therefore, EVs secreted by autologous cells of diabetic patients are not suitable for self-repair. This work hopes that the 3D printing scaffold designed for sustained-release EVs will provide a new strategy for acellular tissue engineering bone repair in diabetic patients.

Injectable hydrogel loaded with 4-octyl itaconate enhances cartilage regeneration by regulating macrophage polarization.

Macrophages play a distinctive role in the early stage of inflammation after cartilage defects. Previous studies have shown that macrophages can express different phenotypes, among which M2 polarization is important to maintain the balance of the inflammatory microenvironment and promote cartilage regeneration. In this study, 4-octyl itaconic acid (4-OI), a derivative of the endogenous metabolite itaconic acid, was used to regulate the polarization behavior of macrophages and enhance cartilage repair. Oxidized sodium alginate (OSA) and gelatin (GEL) were selected as materials to form injectable hydrogels with the function of sustained release of 4-OI. In vivo and in vitro experiments have verified that the OSA/GEL hydrogel system loaded with 4-OI could promote M2 macrophage polarization and inhibit the inflammatory reaction. A rat knee joint cartilage defect model further confirmed its role in promoting cartilage regeneration in the later stage. In this study, the OSA/GEL hydrogel was successfully fabricated as a vehicle for delivering 4-OI, which could evidently alleviate the inflammatory reaction and thus accelerate tissue regeneration. The results of this study provide a new method for promoting subsequent tissue regeneration by regulating the early immune response.

Comparative physiological and metabolomic analyses reveal that Fe3O4 and ZnO nanoparticles alleviate Cd toxicity in tobacco.

BACKGROUND: Heavy metals repress tobacco growth and quality, and engineered nanomaterials have been used for sustainable agriculture. However, the underlying mechanism of nanoparticle-mediated cadmium (Cd) toxicity in tobacco remains elusive. RESULTS: Herein, we investigated the effects of Fe3O4 and ZnO nanoparticles (NPs) on Cd stress in tobacco cultivar 'Yunyan 87' (Nicotiana tabacum). Cd severely repressed tobacco growth, whereas foliar spraying with Fe3O4 and ZnO NPs promoted plant growth, as indicated by enhancing plant height, root length, shoot and root fresh weight under Cd toxicity. Moreover, Fe3O4 and ZnO NPs increased, including Zn, K and Mn contents, in the roots and/or leaves and facilitated seedling growth under Cd stress. Metabolomics analysis showed that 150 and 76 metabolites were differentially accumulated in roots and leaves under Cd stress, respectively. These metabolites were significantly enriched in the biosynthesis of amino acids, nicotinate and nicotinamide metabolism, arginine and proline metabolism, and flavone and flavonol biosynthesis. Interestingly, Fe3O4 and ZnO NPs restored 50% and 47% in the roots, while they restored 70% and 63% in the leaves to normal levels, thereby facilitating plant growth. Correlation analysis further indicated that these metabolites, including proline, 6-hydroxynicotinic acid, farrerol and quercetin-3-O-sophoroside, were significantly correlated with plant growth. CONCLUSIONS: These results collectively indicate that metal nanoparticles can serve as plant growth regulators and provide insights into using them for improving crops in heavy metal-contaminated areas.

Identification and fine mapping of rtms1-D, a gene responsible for reverse thermosensitive genic male sterility from Diannong S-1X.

Thermosensitive genic male sterility (TGMS) has been widely used in two-line hybrid rice breeding. Due to hybrid seed production being highly affected by changeable environments, its application scope is limited to some extent. Thus, it is of great importance to identify potential TGMS genes in specific rice varieties. Here, Diannong S-1 xuan (DNS-1X), a reverse TGMS (RTGMS) japonica male sterile line, was identified from Diannong S-1. Genetic analysis showed that male sterility was tightly controlled by a single recessive gene, which was supported by the phenotype of the F1 and F2:3 populations derived from the cross between DNS-1X and Yunjing 26 (YJ26). Combining simple sequence repeat (SSR) markers and bulked segregation analysis (BSA), we identified a 215 kb region on chromosome 10 as a candidate reverse TGMS region, which was designated as rtms1-D. It was narrower than the previously reported RTGMS genes rtms1 and tms6(t). The fertility conversion detected in the natural environment showed that DNS-1X was sterile below 28-30 °C; otherwise, it was fertile. Histological analysis further indicated that the pollen abortion was occurred in the young microspore stage. This study will provide new resources for two-line hybrid rice and pave the way for molecular breeding of RTGMS lines.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

MYB70 modulates seed germination and root system development in Arabidopsis.

Crosstalk among ABA, auxin, and ROS plays critical roles in modulating seed germination, root growth, and suberization. However, the underlying molecular mechanisms remain largely elusive. Here, MYB70, a R2R3-MYB transcription factor was shown to be a key component of these processes in Arabidopsis thaliana. myb70 seeds displayed decreased sensitivity, while MYB70-overexpressing OX70 seeds showed increased sensitivity in germination in response to exogenous ABA through MYB70 physical interaction with ABI5 protein, leading to enhanced stabilization of ABI5. Furthermore, MYB70 modulates root system development (RSA) which is associated with increased conjugated IAA content and H2O2/O2 ⋅- ratio but reduced root suberin deposition, consequently affecting nutrient uptake. In support of these data, MYB70 positively regulates the expression of auxin conjugation-related GH3, while negatively peroxidase-encoding and suberin biosynthesis-related genes. Our findings collectively revealed a previously uncharacterized component that modulates ABA and auxin signaling pathways, H2O2/O2 ⋅- balance, and suberization, consequently regulating RSA and seed germination.

A hybrid sterile locus leads to the linkage drag of interspecific hybrid progenies.

Interspecific hybridization plays an important role in rice breeding by broadening access to desirable traits such as disease resistance and improving yields. However, interspecific hybridization is often hindered by hybrid sterility, linkage drag, and distorted segregation. To mine for favorable genes from Oryza glaberrima, we cultivated a series of BC4 introgression lines (ILs) of O. glaberrima in the japonica rice variety background (Dianjingyou 1) in which the IL-2769 (BC4F10) showed longer sterile lemmas, wider grains and spreading panicles compared with its receptor parent, suggesting that linkage drag may have occurred. Based on the BC5F2 population, a hybrid sterility locus, S20, a long sterile lemma locus, G1-g, and a new grain width quantitative trait locus (QTL), qGW7, were mapped in the linkage region about 15 centimorgan (cM) from the end of the short arm of chromosome 7. The hybrid sterility locus S20 from O. glaberrima eliminated male gametes of Oryza sativa, and male gametes carrying the alleles of O. sativa in the heterozygotes were aborted completely. In addition, the homozygotes presented a genotype of O. glaberrima, and homozygous O. sativa were not produced. Surprisingly, the linked traits G1-g and qGW7 showed similar segregation distortion. These results indicate that S20 was responsible for the linkage drag. As a large number of detected hybrid sterility loci are widely distributed on rice chromosomes, we suggest that hybrid sterility loci are the critical factors for the linkage drag in interspecific and subspecific hybridization of rice.

Comparative Physiological and Transcriptomic Analyses Reveal the Toxic Effects of ZnO Nanoparticles on Plant Growth.

Zinc oxide (ZnO) nanoparticles (nZnO) are among the most commonly used nanoparticles (NPs), and they have been shown to have harmful effects on plants. However, the molecular mechanisms underlying nZnO tolerance and root sensing of NP stresses have not been elucidated. Here, we compared the differential toxic effects of nZnO and Zn2+ toxicity on plants during exposure and recovery using a combination of transcriptomic and physiological analyses. Although both nZnO and Zn2+ inhibited primary root (PR) growth, nZnO had a stronger inhibitory effect on the growth of elongation zones, whereas Zn2+ toxicity had a stronger toxic effect on meristem cells. Timely recovery from stresses is critical for plant survival. Despite the stronger inhibitory effect of nZnO on PR growth, nZnO-exposed plants recovered from stress more rapidly than Zn2+-exposed plants upon transfer to normal conditions, and transcriptome data supported these results. In contrast to Zn2+ toxicity, nZnO induced endocytosis and caused microfilament rearrangement in the epidermal cells of elongation zones, thereby repressing PR growth. nZnO also repressed PR growth by disrupting cell wall organization and structure through both physical interactions and transcriptional regulation. The present study provides new insight into the comprehensive understanding and re-evaluation of NP toxicity in plants.

The SNAC-A Transcription Factor ANAC032 Reprograms Metabolism in Arabidopsis.

Studies have indicated that the carbon starvation response leads to the reprogramming of the transcriptome and metabolome, and many genes, including several important regulators, such as the group S1 basic leucine zipper transcription factors (TFs) bZIP1, bZIP11 and bZIP53, the SNAC-A TF ATAF1, etc., are involved in these physiological processes. Here, we show that the SNAC-A TF ANAC032 also plays important roles in this process. The overexpression of ANAC032 inhibits photosynthesis and induces reactive oxygen species accumulation in chloroplasts, thereby reducing sugar accumulation and resulting in carbon starvation. ANAC032 reprograms carbon and nitrogen metabolism by increasing sugar and amino acid catabolism in plants. The ChIP-qPCR and transient dual-luciferase reporter assays indicated that ANAC032 regulates trehalose metabolism via the direct regulation of TRE1 expression. Taken together, these results show that ANAC032 is an important regulator of the carbon/energy status that represses photosynthesis to induce carbon starvation.

Comparative physiological responses and transcriptome analysis reveal the roles of melatonin and serotonin in regulating growth and metabolism in Arabidopsis.

BACKGROUND: Melatonin and serotonin are well-known signaling molecules that mediate multiple physiological activities in plants, including stress defense, growth, development, and morphogenesis, but their underlying mechanisms have not yet been thoroughly elucidated. In this study, we investigated the roles of melatonin and serotonin in modulating plant growth and defense by integrating physiological and transcriptome analyses in Arabidopsis. RESULTS: Moderate concentrations of melatonin and serotonin did not affect primary root (PR) growth but markedly induced lateral root (LR) formation. Both melatonin and serotonin locally induced the expression of the cell-wall-remodeling-related genes LBD16 and XTR6, thereby inducing LR development. Our data support the idea that melatonin and serotonin lack any auxin-like activity. Treatment with 50 µM serotonin significantly improved PSII activity, and the transcriptome data supported this result. Melatonin and serotonin slightly affected glycolysis and the TCA cycle; however, they markedly regulated the catabolism of several key amino acids, thereby affecting carbon metabolism and energy metabolism. Melatonin and serotonin improved iron (Fe) deficiency tolerance by inducing Fe-responsive gene expression. CONCLUSIONS: Overall, our results from the physiological and transcriptome analyses reveal the roles of melatonin and serotonin in modulating plant growth and stress responses and provide insight into novel crop production strategies using these two phytoneurotransmitters.

Involvement of reactive oxygen species and auxin in serotonin-induced inhibition of primary root elongation.

The well-known neurotransmitter 5-hydroxytryptamine (serotonin) not only regulates sleep and mood in humans and animals but may also play important roles in modulating growth, development, and defense responses, such as seed germination, flowering, and abiotic stress tolerance, in plants. Serotonin inhibits primary root (PR) growth; however, the physiological and molecular mechanisms underlying serotonin-mediated PR growth inhibition remain largely unclear. Here, we investigate the effects of serotonin on root growth and development in Arabidopsis. Serotonin inhibits PR elongation by affecting both the meristem and elongation zones. In the meristem zone, serotonin represses both meristem cell division potential and stem cell niche activity. Serotonin induces H2O2 overaccumulation in the elongation zone and reduces O2- accumulation in the meristem zone by a UPB1 pathway, thereby disrupting reactive oxygen species (ROS) equilibrium in root tips, thus resulting in PR growth inhibition. Serotonin also regulates auxin distribution in root tips by decreasing auxin-related gene expression and repressing auxin transport through modulation of AUX1 and PIN2 abundances in root tips. Taken together, our data indicate that high concentrations of serotonin result in stress responses in plants by inhibiting PR elongation through the regulation of H2O2 and O2- distribution in PR tips and through an auxin pathway via the repression of auxin biosynthesis and transport.

UV-B Radiation Induces Root Bending Through the Flavonoid-Mediated Auxin Pathway in Arabidopsis.

Ultraviolet (UV)-B radiation-induced root bending has been reported; however, the underlying mechanisms largely remain unclear. Here, we investigate whether and how auxin and flavonoids are involved in UV-B radiation-induced root bending in Arabidopsis using physiological, pharmacological, and genetic approaches. UV-B radiation modulated the direction of root growth by decreasing IAA biosynthesis and affecting auxin distribution in the root tips, where reduced auxin accumulation and asymmetric auxin distribution were observed. UV-B radiation increased the distribution of auxin on the nonradiated side of the root tips, promoting growth and causing root bending. Further analysis indicated that UV-B induced an asymmetric accumulation of flavonoids; this pathway is involved in modulating the accumulation and asymmetric distribution of auxin in root tips and the subsequent redirection of root growth by altering the distribution of auxin carriers in response to UV-B radiation. Taken together, our results indicate that UV-B radiation-induced root bending occurred through a flavonoid-mediated phototropic response to UV-B radiation.

Lanthanum Inhibits Primary Root Growth by Repressing Auxin Carrier Abundances in Arabidopsis.

Lanthanum (La) is one of rare earth elements that was used as a crop growth stimulants; however, high concentration of La markedly inhibited plant growth. Our previous study indicated that, although La induced the expression of auxin biosynthesis-related genes, it markedly repressed primary root (PR) elongation by reducing auxin accumulation in PR tips. In this study, we exhibited that La reduces the abundances of auxin carriers. Treatment with La markedly inhibited the auxins IAA-, 2,4-D-, and NAA-induced elevation of DR5:GUS activity in the roots, suggesting that La inhibited auxin transport through both the influx and efflux transporters. Supplementation with auxin transport inhibitor naphthylphthalamic acid in La-treated seedlings did not further reduce PR growth compared with that of the La treatment alone, further confirmed that auxin transport is involved in La-induced inhibition of PR growth. Analysis of the protein abundances using the transgenic AUX1-YFP and PIN1/2/4/7-GFP marker lines indicated that La treatment reduced the abundances of all these auxin carriers in the PR tips. La also increased the stabilization of Aux/IAA protein AXR3. Taken together, these results indicated that La treatment inhibits PIN-mediated auxin transport and subsequently impairs auxin distribution and PR growth via reducing auxin carrier abundances.

Nectar robbers pollinate flowers with sexual organs hidden within corollas in distylous Primula secundiflora (Primulaceae).

Nectar robbers are thought rarely to pollinate flowers, especially those with sexual organs hidden within corollas. In this study, we examined whether robbers pollinate flowers of distylous Primula secundiflora. Distylous plants have two floral morphs. Pin flowers have long styles and short stamens, and thrum flowers have short styles and long stamens. Flowers of P. secundiflora were commonly robbed by bumble-bees, and robbing holes were always situated between high and low sexual organs for both floral morphs. We observed that pollen grains of pin flowers were removed while thrum flowers received fresh pollen grains immediately after flowers were robbed. We manipulated flowers so that only nectar robbers could visit them. This resulted in 98 per cent of thrum flowers and 6 per cent of pin flowers setting fruit, and seed number per thrum fruit was also significantly higher than per pin fruit. Our findings suggest that nectar robbers transfer pollen from pin flowers to thrum flowers effectively, and consequently increase male fitness of the pin morph and female fitness of the thrum morph. Such asymmetrical pollen flow caused by nectar robbers may act as an important selective agent in floral fitness and evolution of distyly.