Plant Pollen Grains:A Move Towards Green Drug and Vaccine Delivery Systems

Pollen grains and plant spores have emerged as innovative biomaterials for various applications such as drug/vaccine delivery,catalyst support,and the removal of heavy metals.The natural microcapsules comprising spore shells and pollen grain are designed for protecting the genetic materials of plants from exterior impairments.Two layers make up the shell,the outer layer(exine)that comprised largely of sporopollenin,and the inner layer(intine)that built chiefly of cellulose.These microcapsule shells,namely hollow sporopollenin exine capsules have some salient features such as homogeneity in size,non-toxic nature,resilience to both alkalis and acids,and the potential to withstand at elevated temperatures;they have displayed promising potential for the microencapsulation and the controlled drug delivery/release.The important attribute of mucoadhesion to intestinal tissues can prolong the interaction of sporopollenin with the intestinal mucosa directing to an augmented effectiveness of nutraceutical or drug delivery.Here,current trends and prospects related to the application of plant pollen grains for the delivery of vaccines and drugs and vaccine are discussed.

Fabrication and Characterisation of Novel Natural Lycopodium clavatum Sporopollenin Microcapsules Loaded In-Situ with Nano-Magnetic Humic Acid-Metal Complexes

Sporopollenin exines microcapsules, derived from the naturally occurring spores of Lycopodium clavatum, have been loaded in-situ with humic acid sodium salt-Zinc (HA-Zn) complex. The chemical treatment method utilised to prepare the sporopollenin microcapsules from raw spores was discussed and the resulted sporopollenin microcapsules were characterised using SEM, TGA and FTIR. Metal complexes of the sodium salt of humic acid and zinc ion were prepared using different protocols and in-situ loaded into the pre-treated sporopollenin microcapsules. The resulted complex was characterised before and after the encapsulation process using FTIR, TGA and XRD techniques. The morphology of the empty and loaded sporopollenin was not altered. Infrared spectroscopy revealed an increase in the absorption for COO– vibrations at 1583 and 1384 cm–1 in the FTIR spectra of HA-Zn complex compared to that of the original sodium salt of humic acid, indicative of bonding of the metal ions in hydrated form to the carboxyl or phenolic hydroxyl groups or both of the sodium humate molecules. TGA results of the HA-Zn complex loaded sporopollenin showed that around %15 of residual HA-Zn was successfully encapsulated indicative of the efficiency of the protocol used. We showed also that biodegradable magnetite nanoparticles can be surface modified with HA and encapsulated into sporopollenin. The resulted biosorbents microcapsules can be used for enhanced magnetic removal of either heavy metals or HA from different aqueous media.

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin

Sporopollenin in the pollen cell wall protects male gametophytes from stresses.Phenylpropanoid derivatives,including guaiacyl(G)lignin units,are known to be structural components of sporopollenin,but the exact composition of sporopollenin remains to be fully resolved.We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance(NMR)and gas chromatography–mass spectrometry(GC–MS).The NMR and GC–MS results confirmed the presence of p-hydroxyphenyl(H),G,and syringyl(S)lignin units in sporopollenin from maize and Arabidopsis.Strikingly,H units account for the majority of lignin monomers in sporopollenin from these species.We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein(ZmVAMP726)that is strongly associated with lignin monomer composition of maize sporopollenin.Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis,indicating that VAMP726 is functionally conserved in monocot and dicot plants.Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726,which affects sporopollenin composition and stress resistance in pollen.

Atomic force microscopic observation on substructure of pollen exine in Cedrus deodara and Metasequoia glyptostroboides

The substructure of pollen exine in Cedrus deodara (Roxb.) Loud, and Metasequoia glyptostroboides Hu et Cheng has been examined with an atomic force microscope (AFM). The results indicate that the exine substructure units containing sporopollenin in two species are similar in shape, which are granular, but slightly different in size. In Cedrus the substructure unit of pollen exine appears to be 56-99 nm long and 42-74 nm wide, while in Metasequoia it appears to be 81-118 nm long and 43-98 nm wide. It has been observed that the subunits of pollen exine in Cedrus arranged tightly to form short-rod-like or spheroidal pollen exine units, several or more than ten of which formed an island-like structure. There are various spaces among these island-like structures which are interconnected to occupy the entire pollen exine. In Metasequoia, the subunits of pollen exine also arranged tightly with a distribution tendency of cluster of 3-10, however, no obvious boundary exists among these clusters. From our

Pollen wall pattern in Arabidopsis

The pollen wall is a solid and variously sculptured structure. This pattern is determined inside a tetrad. During meiosis, the callose wall is formed outside of the meiocyte/microspore to form a tetrad. Then, primexine is deposited between the callose wall and the microspore plasma membrane which will become undulated. The sporopollenin deposits on top of the undulated membrane and develops into the pollen wall pattern, while the callose wall is gradually degraded. In recent years, much progress has been made in the study of pollen wall pattern formation, at both molecular and genetic levels. In this review,we summarize these achievements mainly in Arabidopsis.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants

The outer wall of pollen and spores,namely the exine,is composed of sporopollenin,which is highly resistant to chemical reagents and enzymes.In this study,we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants.Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin.Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives,including p-hydroxybenzoate(p-BA),p-coumarate(p-CA),ferulate(FA),and lignin guaiacyl(G)units.The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis,consistent with the fact that the sporopollenin precursor originates from the tapetum.Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation.In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata,phenylpropanoid derivatives including p-BA and p-CA were also detected,but G units were not.Taken together,our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants.In addition,sporopollenin autofluorescence spectra of bryophytes,such as Physcomitrella and Haplocladium,exhibit distinct characteristics compared with those of vascular plants,indicating the diversity of sporopollenin among land plants.

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton

The double-recessive genic male-sterile(ms)line ms5 ms6 has been used to develop cotton(Gossypium hirsutum)hybrids for many years,but its molecular-genetic basis has remained unclear.Here,we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing.Ms5 and Ms6 are highly expressed in stages 7–9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D.The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312(GhCYP703A2-A^(312aa)),and ms6 carries three nonsynonymous substitutions(D98E,E168K,and G198R)and a synonymous mutation(L11L).Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids,and the ms5(GhCYP703A2-A^(312aa))and ms6(GhCYP703A2-D^(D98E,E168K,G198R))mutant proteins have decreased enzyme activities.Biochemical and lipidomic analyses showed that in ms5 ms6 plants,C12–C18 free fatty acid and phospholipid levels are significantly elevated in stages 7–9 anthers,while stages 8–10 anthers lack sporopollenin fluorescence around the pollen,causing microspore degradation and male sterility.Overall,our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility,providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.

NERD1 is required for primexine formation and plasma membrane undulation during microsporogenesis in Arabidopsis thaliana

The primexine formation and plasma membrane undulation are the crucial steps of pollen wall formation in many angiosperms.However,the molecular mechanism underlining these processes is largely unknown.In Arabidopsis,NEW ENHANCER OF ROOT DWARFISM1(NERD1),a transmembrane protein,was reported to play pleiotropic roles in plant development including male fertility control;while,how NERD1 disruption impacts male reproduction is yet unclear.Here,we revealed that the male sterility of nerd1 mutants is attributed to defects in early steps of pollen wall formation.We found that nerd1-2 is void of primexine formation and microspore plasma membrane undulation,defective in callose deposition.Consequently,sporopollenin precursors are unable to deposit and assemble on the microspore surface,but instead accumulated in the anther locule and tapetal cells,and ultimately leading to microspore abortion.NERD1 is localized in the Golgi and is expressed in both vegetative and reproductive organs,with the highest expression in reproductive tissues,including the tapetum,male meiocytes,tetrads and mature pollen grains.Our results suggest that NERD1 is required for the primexine deposition and microspore plasma membrane undulation,thus essential for sporopollenin assembly and pollen exine formation.