Histochemical and ultrastructural analysis of tapetum and sporoderm development in relation to precocious pollenkitt production of Garcinia dulcis (Roxb.) Kurz.

Garcinia dulcis (Roxb.) Kurz (Clusiaceae) is a medicinal plant native to Southeastern Asia, with a peculiar, precocious pollenkitt production in early microspore development. We aimed to find out whether different secretory activities of the tapetum or a premature sporoderm development provides additional evidence for our recent hypothesis for the precocious pollenkitt production. Histology, histochemistry and ultrastructure of tapetum and sporoderm development during pollenkitt secretion in Garcinia dulcis were conducted, based on light and electron microscopy analysis. The results showed that Garcinia dulcis possesses normal pollen development. The presence of two different pollen coating types, precocious pollenkitt (L1) and common pollenkitt (L2), in the anther tapetum indicate that they are produced in two different active stages of the secretory tapetum. The precocious pollenkitt production and transport to the locule takes place in early active tapetal cells at early tetrad to early microspore stage and is ongoing until late microspore stage. The production of the second type of pollenkitt (L2) starts shortly after the first active tapetum stage together with the formation of sporopollenin precursors. The sporoderm formation was completed at late microspore stage, when the tapetal cell walls start to disintegrate. Orbicules are lining the inner tapetum wall at middle to late microspore stage. ER (during early microspore stage) and plastids (during late microspore stage) were the two main sources of pollenkitt, which finally fused to pollenkitt droplets when the tapetal cells degenerated at mature bicellular pollen stage.

An Ecofriendly Nature-Inspired Microcarrier for Enhancing Delivery, Stability, and Biocidal Efficacy of Phage-Based Biopesticides.

In pursuit of sustainable agricultural production, the development of environmentally friendly and effective biopesticides is essential to improve food security and environmental sustainability. Bacteriophages, as emerging biocontrol agents, offer an alternative to conventional antibiotics and synthetic chemical pesticides. The primary challenges in applying phage-based biopesticides in agricultural settings are their inherent fragility and low biocidal efficacy, particularly the susceptibility to sunlight exposure. This study addresses the aforementioned challenges by innovatively encapsulating phages in sporopollenin exine capsules (SECs), which are derived from plant pollen grains. The size of the apertures on SECs could be controlled through a non-thermal and rapid process, combining reinflation and vacuum infusion techniques. This unique feature facilitates the high-efficiency encapsulation and controlled release of phages under various conditions. The proposed SECs could encapsulate over 9 log PFU g-1 of phages and significantly enhance the ultraviolet (UV) resistance of phages, thereby ensuring their enhanced survivability and antimicrobial efficacy. The effectiveness of SECs encapsulated phages (T7@SECs) in preventing and treating bacterial contamination on lettuce leaves is further demonstrated, highlighting the practical applicability of this novel biopesticide in field applications. Overall, this study exploits the potential of SECs in the development of phage-based biopesticides, presenting a promising strategy to enhancing agricultural sustainability.

Systematic Implications of Palynomorph Diversity Using Microscopic Trends Among Asteraceous Flora From the Drylands of Baluchistan, Pakistan.

Pollen micromorphological traits with taxonomic implications are first reported from the study area for 50 Asteraceous taxa belonging to nine tribes. Cichorieae (21 taxa), Cardueae (11 taxa), Inuleae (six taxa), and Anthemideae (four taxa) are the leading tribes. The research included Cousinia haeckeliae, Himalaiella afghana, Pterachaenia stewartii (endemic to Afghanistan and Pakistan), and Xylanthemum macropodum (endemic to Baluchistan). Light and scanning electron microscopy were employed for the visualization of pollen photomicrographs. The data was analyzed statistically via SPSS, PAST, and Origin. Significant diagnostic qualitative and quantitative palynological traits were explored for discrimination down to the species level within the tribes. All the investigated taxa possessed radial symmetry, isopolarity, and monad form (characters for distinction at the subdivision level). The aperture types were trizonocolporate, tetrazonocolporate, and tricolporate with number position and character (NPC) formulas N3P4C5, N4P4C5, and N3P4C3. Goniotreme, peritreme, and ptychotreme types of amb were recognized. Echinate, echinate lophate, scabrate, and gemmate sculpturing were present with and without perforated surface patterns. Variations in the shapes in polar and equatorial views and lacuna shapes further assisted the separation of taxa. The observed shape classes were perprolate, prolate spheroidal, prolate, subprolate, oblate spheroidal, suboblate, and oblate. Principal component analysis, correlation, standard probability plots, and ridge line paired features plot for quantitative variables determined the positive correlation between the length and width of colpi in equatorial and polar view with polar axis and equatorial diameter and number of spines between colpi with the number of spines per pollen. The number of spines per pollen was negatively correlated with the width and length of colpi in the polar view. Multiple sample analysis of variance (ANOVA) concluded that a high statistically significant difference exists among the means of analyzed traits. The examined qualitative and quantitative palynological traits revealed noticeable variations, thus providing the source for species discrimination in Asteraceous tribes.

OsALKBH9-mediated m6A demethylation regulates tapetal PCD and pollen exine accumulation in rice.

The N6-methyladenosine (m6A) mRNA modification is crucial for plant development and stress responses. In rice, the male sterility resulting from the deficiency of OsFIP37, a core component of m6A methyltransferase complex, emphasizes the significant role of m6A in male fertility. m6A is reversible and can be removed by m6A demethylases. However, whether mRNA m6A demethylase regulates male fertility in rice has remained unknown. Here, we identify the mRNA m6A demethylase OsALKBH9 and demonstrate its involvement in male fertility regulation. Knockout of OsALKBH9 causes male sterility, dependent on its m6A demethylation activity. Cytological analysis reveals defective tapetal programmed cell death (PCD) and excessive accumulation of microspores exine in Osalkbh9-1. Transcriptome analysis of anthers shows up-regulation of genes involved in tapetum development, sporopollenin synthesis, and transport pathways in Osalkbh9-1. Additionally, we demonstrate that OsALKBH9 demethylates the m6A modification in TDR and GAMYB transcripts, which affects the stability of these mRNAs and ultimately leads to excessive accumulation of pollen exine. Our findings highlight the precise control of mRNA m6A modification and reveal the pivotal roles played by OsALKBH9-mediated m6A demethylation in tapetal PCD and pollen exine accumulation in rice.

Exploring microscopic pollen morphology in herbaceous Flora: Insights and analysis using scanning electron microscopy.

Microscopic techniques can be applied to solve taxonomic problems in the field of plant systematic and are extremely versatile in nature. This study was focused on the new approaches to visualizing the imaging, tool to cover the micro-structural techniques applied to the pollen study of flowers. The current research was proposed to evaluate microscopic pollen morphological attributes using light and scanning electron microscopy of herbaceous flora from Samarkand, Uzbekistan. A total of 13 herbaceous species, classified into 11 different families were collected, pressed, and identified, and then acetolyzed their pollen to visualize under light and scanning electron microscopy. Herbaceous flora can be characterized by small to very large-sized pollen morphotypes presenting four types of pollen shapes, prolate spheroidal (six species), spheroidal (three species) and prolate and oblate (two species each). The polar diameter and equatorial distance were calculated maximum in Hibiscus syriacus 110.55 and 111.2 µm respectively. Pollen of six different types was found namely tricolporate pollen observed in seven species, tricolpate and pantoporate in two species each, sulcate in Gagea olgae and hexacolpate pollen was examined in Salvia rosmarinus. Exine ornamentation of pollen was examined tectate perforate, verrucate-reticulate, micro-reticulate, reticulate, reticulate-cristatum, gemmate-echinate, echinate-perforate, perforate-striate, rugulate, rugulate-striate, bi-reticulate, reticulate-perforate and perforate-micro-reticulate showing great variations. Exine thickness was noted highest for Rosa canina 2.9 µm and minimum in Punica granatum 0.65 µm. This study of pollen imaging visualization of herbaceous flora contributes to the opportunity for the taxonomic evaluation of and fills knowledge gaps in studies of herbaceous flora identification using classical microscopic taxonomic tools for their accurate identification. RESEARCH HIGHLIGHTS: Pollen in unexplored herbaceous flora of the Samarkand region was studied with light and scanning electron microscopic pollen study. There is a high variation in observed pollen micromorphological characters. Pollen microscopic morphology has important taxonomic value for the identification of herbaceous species.

Pollen morphology of Acanthoideae species from the Shivalik Foothills in Rajaji National Park (Uttarakhand, India).

Apart from its role in plant taxonomy, the understanding of pollen morphology is considered an essential interdisciplinary tool in diverse applied fields, including plant systematics, melissopalynology, aeropalynology, forensic palynology, paleopalynology, and copropalynology. In palynotaxonomy, it is frequently employed to classify and validate natural groups across a range of hierarchical levels, from higher categories down to the infrageneric level. The subfamily Acanthoideae, within the Acanthaceae family, consists of a diverse group of flowering plants that are distributed globally. The present study attempted a comprehensive analysis of the pollen morphology, employing both light microscopy (LM) and scanning electron microscopy (SEM), for a total of 13 Acanthoideae species from the Shivalik Foothills in Rajaji National Park (located in the northern Indian state of Uttarakhand, Western Himalaya). The findings indicated that the Acanthoideae is characterized by eurypalynous features, and the studied species exhibited pollen grains that were monads, radially symmetrical, and varied in size from small to large. The pollen grains were predominantly tricolporate or heteroaperturate, with porate occurrences being rare. The significant variation in exine sculpturing, including reticulate, coarsely reticulate, and bireticulate patterns, holds substantial taxonomic significance. The detailed presentation encompasses pollen morphological characters described with LM and SEM micrographs, along with a species-level identification key. RESEARCH HIGHLIGHTS: This article provides a thorough analysis of the pollen morphology of Acanthoideae taxa using both light microscopy and scanning electron microscopy, covering 13 species across 10 genera and revealing a broad spectrum of pollen characteristics, including size, shape, aperture type, and exine sculpturing. The microscopic investigation of these Acanthoideae species not only enhances our understanding of their pollen morphology but also aids in species identification through the development of a pollen-based key.

A subclass II bHLH transcription factor in Marchantia polymorpha gives insight into the ancestral land plant trait of spore formation.

Sporopollenin is often said to be one of the toughest biopolymers known to man. The shift in dormancy cell wall deposition from around the diploid zygotes of charophycean algae to sporopollenin around the haploid spores of land plants essentially imparted onto land plants the gift of passive motility, a key acquisition that contributed to their vast and successful colonization across terrestrial habitats.1,2 A putative transcription factor controlling the land plant mode of sporopollenin deposition is the subclass II bHLHs, which are conserved and novel to land plants, with mutants of genes in angiosperms and mosses divulging roles relating to tapetum degeneration and spore development.3,4,5,6,7 We demonstrate that a subclass II bHLH gene, MpbHLH37, regulates sporopollenin biosynthesis and deposition in the model liverwort Marchantia polymorpha. Mpbhlh37 sporophytes show a striking loss of secondary wall deposits of the capsule wall, the elaters, and the spore exine, all while maintaining spore viability, identifying MpbHLH37 as a master regulator of secondary wall deposits of the sporophyte. Localization of MpbHLH37 to the capsule wall and elaters of the sporophyte directly designates these tissue types as a bona fide tapetum in liverworts, giving support to the notion that the presence of a tapetum is an ancestral land plant trait. Finally, as early land plant spore walls exhibit evidence of tapetal deposition,8,9,10,11,12 a tapetal capsule wall could have provided these plants with a developmental mechanism for sporopollenin deposition.

Characterization and expression analysis of chalcone synthase gene family members suggested their roles in the male sterility of a wheat temperature-sensitive genic male sterile (TGMS) line.

Chalcone synthase (CHS), also known as the plants-specific type III polyketide synthases (PKSs), catalyzes the first key step in the biosynthesis of plant flavonoids. Flavonoids are one of the most important secondary metabolites which participate in flower pigmentation and pollen fertility. Recent reports have demonstrated the role of the CHS family in plant pollen exine formation. This study focused on the potential roles of CHS in the pollen exine formation of wheat. In the present study, a genome-wide investigation of the CHS family was carried out, and 87 CHS genes were identified in wheat. TaCHS3, TaCHS10, and TaCHS13 are wheat orthologs of Arabidopsis LESS ADHESIVE POLLEN (LAP5); TaCHS58, TaCHS64, and TaCHS67 are wheat orthologs of AtLAP6. TaCHS3, TaCHS10, and TaCHS67 showed anther-specific patterns. The expression of TaCHS3, TaCHS10, and TaCHS67 was positively co-expressed with sporopollenin biosynthetic genes, including TaCYP703A2, TaCYP704B1, TaDRL1, TaTKPR2, and TaMS2. Coincidently, the expression of TaCHS3, TaCHS10, and TaCHS67, together with those sporopollenin biosynthetic genes, were repressed at the tetrads and uninucleate stages in the temperature-sensitive genic male-sterile (TGMS) line BS366 under sterile conditions. Wheat anther-specific CHS genes might participate in the exine formation of BS366 through co-expressing with sporopollenin biosynthetic genes, which will undoubtedly provide knowledge of the roles of CHS in wheat pollen development.

OsTKPR1 proteins with a single amino acid substitution fail the synthesis of a specific sporopollenin precursor and cause abnormal exine and pollen development in rice.

Fatty acid derivatives are key components of rice pollen exine. The synthesis of aliphatic sporopollenin precursors are initiated in the plastids of the tapetal cells, followed by multiple-step reactions conducted in the endoplasmic reticulum (ER). However, the relative contribution of different precursors to the precise structure of sporopollenin remains largely elusive, let alone the underlying mechanism. Here, we report that two complete male sterile mutants ostkpr1-3 (Tetraketide α-pyrone reductase 1-3, with OsTKPR1P124S substitution) and ostkpr1-4 (with truncated OsTKPR1stop) are defective in pollen exine, Ubisch body and anther cuticle development where ostkpr1-4 display severer phenotypes. Remarkably, OsTKPR1 could produce reduced hydroxylated tetraketide α-pyrone and reduced tetraketide α-pyrone, whereas OsTKPR1P124S fails to produce the latter. Pairwise interaction assays show that mutated OsTKPR1P124S is able to integrate into a recently characterized metabolon, thus its altered catalytic activity is not due to dis-integrity of the metabolon. In short, we find that reduced tetraketide α-pyrone is a key sporopollenin precursor required for normal exine formation, and the conserved 124th proline of OsTKPR1 is essential for the reduction activity. Therefore, this study provided new insights into the sporopollenin precursor constitution critical for exine formation.

Multifaceted roles of pollen in the management of cancer.

Oral drug delivery of microparticles demonstrates shortcomings like aggregation, decreased loading capacity and batch-to-batch variation, which limits its scale-up. Later, porous structures gained attention because of their large surface-to-volume ratio, high loading capacity and ability to carry biomacromolecules, which undergo degradation in GIT. But there are pitfalls like non-uniform particle size distribution, the impact of porogen properties, and harsh chemicals. To circumvent these drawbacks, natural carriers like pollen are explored in drug delivery, which withstands harsh environments. This property helps to subdue the acid-sensitive drug in GIT. It shows uniform particle size distribution within the species. On the other side, they contain phytoconstituents like flavonoids and polysaccharides, which possess various pharmacological applications. Therefore, pollen has the capability as a carrier system and therapeutic agent. This review focuses on pollen's microstructure, composition and utility in cancer management. The extraction strategies, characterisation techniques and chemical structure of sporopollenin exine capsule, its use in the oral delivery of antineoplastic drugs, and emerging cancer treatments like photothermal therapy, immunotherapy and microrobots have been highlighted. We have mentioned a note on the anticancer activity of pollen extract. Further, we have summarised the regulatory perspective, bottlenecks and way forward associated with pollen.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development.

Because of its significance for plant male fertility and, hence, direct impact on crop yield, pollen exine development has inspired decades of scientific inquiry. However, the molecular mechanism underlying exine formation and thickness remains elusive. In this study, we identified that a previously unrecognized repressor, ZmMS1/ZmLBD30, controls proper pollen exine development in maize. Using an ms1 mutant with aberrantly thickened exine, we cloned a male-sterility gene, ZmMs1, which encodes a tapetum-specific lateral organ boundary domain transcription factor, ZmLBD30. We showed that ZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7, and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine. This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum, and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis. Collectively, these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

Sporogenesis in Physcomitrium patens: Intergenerational collaboration and the development of the spore wall and aperture.

Although the evolution of spores was critical to the diversification of plants on land, sporogenesis is incompletely characterized for model plants such as Physcomitrium patens. In this study, the complete process of P. patens sporogenesis is detailed from capsule expansion to mature spore formation, with emphasis on the construction of the complex spore wall and proximal aperture. Both diploid (sporophytic) and haploid (spores) cells contribute to the development and maturation of spores. During capsule expansion, the diploid cells of the capsule, including spore mother cells (SMCs), inner capsule wall layer (spore sac), and columella, contribute a locular fibrillar matrix that contains the machinery and nutrients for spore ontogeny. Nascent spores are enclosed in a second matrix that is surrounded by a thin SMC wall and suspended in the locular material. As they expand and separate, a band of exine is produced external to a thin foundation layer of tripartite lamellae. Dense globules assemble evenly throughout the locule, and these are incorporated progressively onto the spore surface to form the perine external to the exine. On the distal spore surface, the intine forms internally, while the spiny perine ornamentation is assembled. The exine is at least partially extrasporal in origin, while the perine is derived exclusively from outside the spore. Across the proximal surface of the polar spores, an aperture begins formation at the onset of spore development and consists of an expanded intine, an annulus, and a central pad with radiating fibers. This complex aperture is elastic and enables the proximal spore surface to cycle between being compressed (concave) and expanded (rounded). In addition to providing a site for water intake and germination, the elastic aperture is likely involved in desiccation tolerance. Based on the current phylogenies, the ancestral plant spore contained an aperture, exine, intine, and perine. The reductive evolution of liverwort and hornwort spores entailed the loss of perine in both groups and the aperture in liverworts. This research serves as the foundation for comparisons with other plant groups and for future studies of the developmental genetics and evolution of spores across plants.

Microspore-expressed SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen development in wheat.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermosensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

OsTKPR2 is part of a sporopollenin-producing metabolon required for exine formation in rice.

The sporopollenin polymer is a major component of the pollen exine. Fatty acid derivatives synthesized in the tapetum are among the precursors of sporopollenin. Progress has been made to understand sporopollenin metabolism in rice; however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share a similar expression pattern, and their coding proteins have a similar subcellular localization and enzyme activities towards reduced tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1pro:OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, the anther cuticle, Ubisch bodies, and pollen development were affected in the ostkpr2-1 mutant, where a thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed.

The ZmMYB84-ZmPKSB regulatory module controls male fertility through modulating anther cuticle-pollen exine trade-off in maize anthers.

Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen-stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade-off of lipid-metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male-sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map-based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)-localized polyketide synthase (PKS) with catalytic activities to malonyl-CoA and midchain-fatty acyl-CoA to generate triketide and tetraketide α-pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9-10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84-ZmPKSB regulatory module controlled a trade-off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine-tuning regulation of lipid-metabolic balance to precisely promote anther cuticle and pollen exine formation in plants.

Maize sterility gene DRP1 encodes a desiccation-related protein that is critical for Ubisch bodies and pollen exine development.

Desiccation tolerance is a remarkable feature of pollen, seeds, and resurrection-type plants. Exposure to desiccation stress can cause sporophytic defects, resulting in male sterility. Here, we report the novel maize sterility gene DRP1 (Desiccation-Related Protein 1), which was identified by bulked-segregant analysis sequencing and encodes a desiccation-related protein. Loss of function of DRP1 results in abnormal Ubisch bodies, defective tectum of the pollen exine, and complete male sterility. Our results suggest that DRP1 may facilitate anther dehydration to maintain appropriate water status. DRP1 is a secretory protein that is specifically expressed in the tapetum and microspore from the tetrad to the uninucleate microspore stage. Differentially expressed genes in drp1 are enriched in Gene Ontology terms for pollen exine formation, polysaccharide catabolic process, extracellular region, and response to heat. In addition, DRP1 is a target of selection that appears to have played an important role in the spread of maize from tropical/subtropical to temperate regions. Taken together, our results suggest that DRP1 encodes a desiccation-related protein whose loss of function causes male sterility. Our findings provide a potential genetic resource that may be used to design crops for heterosis utilization.

Grass-specific ABERRANT MICROSPORE DEVELOPMENT 1 is required for maintaining pollen fertility in rice.

Pollen development includes a series of biological events that require precise gene regulation. Although several transcription factors (TFs) have been shown to play roles in maintaining pollen fertility, the major regulatory networks underlying tapetum development and pollen wall formation are largely unknown. Herein, we report that ABERRANT MICROSPORE DEVELOPMENT1 (AMD1), a protein annotated previously as unknown protein, is required for tapetum development and pollen exine patterning in rice (Oryza sativa L.). AMD1 encodes a grass-specific protein exhibiting transactivation activity in the nucleus and is spatiotemporally expressed in the tapetum and microspores during pollen development. Further biochemical assays indicate that AMD1 directly activates the transcription of DEFECTIVE POLLEN WALL (DPW) and POLYKETIDE SYNTHASE2 (OsPKS2), which are both implicated in sporopollenin biosynthesis during exine formation. Additionally, AMD1 directly interacts with TAPETUM DEGENERATION RETARDATION (TDR), a key TF involved in the regulation of tapetum degradation and exine formation. Taken together, we demonstrate that AMD1 is an important regulatory component involved in the TDR-mediated regulatory pathway to regulate sporopollenin biosynthesis, tapetum degradation, and exine formation for pollen development. Our work provides insights into the regulatory network of rice sexual reproduction and a useful target for genetic engineering of new male-sterile lines for hybrid rice breeding.

Hydroxyproline-O-Galactosyltransferases Synthesizing Type II Arabinogalactans Are Essential for Male Gametophytic Development in Arabidopsis.

In flowering plants, male reproductive function is determined by successful development and performance of stamens, pollen grains, and pollen tubes. Despite the crucial role of highly glycosylated arabinogalactan-proteins (AGPs) in male gamete formation, pollen grain, and pollen tube cell walls, the underlying mechanisms defining these functions of AGPs have remained elusive. Eight partially redundant Hyp-galactosyltransferases (named GALT2-GALT9) genes/enzymes are known to initiate Hyp-O-galactosylation for Hyp-arabinogalactan (AG) production in Arabidopsis thaliana. To assess the contributions of these Hyp-AGs to male reproductive function, we used a galt2galt5galt7galt8galt9 quintuple Hyp-GALT mutant for this study. Both anther size and pollen viability were compromised in the quintuple mutants. Defects in male gametogenesis were observed in later stages of maturing microspores after meiosis, accompanied by membrane blebbing and numerous lytic vacuoles. Cytological and ultramicroscopic observations revealed that pollen exine reticulate architecture and intine layer development were affected such that non-viable collapsed mature pollen grains were produced, which were devoid of cell content and nuclei, with virtually no intine. AGP immunolabeling demonstrated alterations in cell wall architecture of the anther, pollen grains, and pollen tube. Specifically, the LM2 monoclonal antibody (which recognized ß-GlcA epitopes on AGPs) showed a weak signal for the endothecium, microspores, and pollen tube apex. Pollen tube tips also displayed excessive callose deposition. Interestingly, expression patterns of pollen-specific AGPs, namely AGP6, AGP11, AGP23, and AGP40, were determined to be higher in the quintuple mutants. Taken together, our data illustrate the importance of type-II AGs in male reproductive function for successful fertilization.

The methyl-CpG-binding domain family member PEM1 is essential for Ubisch body formation and pollen exine development in rice.

Pollen exine is composed of finely-organized nexine, bacula and tectum, and is crucial for pollen viability and function. Pollen exine development involves a complicated molecular network that coordinates the interaction between pollen and tapetal cells, as well as the biosynthesis, transport and assembly of sporopollenin precursors; however, our understanding of this network is very limited. Here, we report the roles of PEM1, a member of methyl-CpG-binding domain family, in rice pollen development. PEM1 expressed constitutively and, in anthers, its expression was detectable in tapetal cells and pollen. This predicted PEM1 protein of 240 kDa had multiple epigenetic-related domains. pem1 mutants exhibited abnormal Ubisch bodies, delayed exine occurrence and, finally, defective exine, including invisible bacula, amorphous and thickened nexine and tectum layer structures, and also had the phenotype of increased anther cuticle. The mutation in PEM1 did not affect the timely degradation of tapetum. Lipidomics revealed much higher wax and cutin contents in mutant anthers than in wild-type. Accordingly, this mutation up-regulated the expression of a set of genes implicated in transcriptional repression, signaling and diverse metabolic pathways. These results indicate that PEM1 mediates Ubisch body formation and pollen exine development mainly by negatively modulating the expression of genes. Thus, the PEM1-mediated molecular network represents a route for insights into mechanisms underlying pollen development. PEM1 may be a master regulator of pollen exine development.

Sol-gel synthesis of TiO2 on Co3O4-coated sporopollenin exine microcapsules (SECs) and photocatalytic performance of new semiconductor heterojunction material.

In this study, a new approach was developed to prepare mesoporous hybrid TiO2/Co3O4 coated on Juglans sporopollenin exine microcapsules (SECs). TiO2 was synthesized on Co3O4-coated SECs used as substrate, by sol-gel method. The obtained semiconductor/semiconductor hetero-junction hybrid materials were characterized with X-ray diffractometry (XRD), UV-Vis absorption spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), particle size distribution, specific surface area, and zeta potential measurements. Photocatalytic performances of hybrid materials were tested for Reactive Black 5 dye under both UV and visible light. Equilibrium pH of the solution containing 10 mg/L Reactive Black 5 dye and 0.1% wt/v TiO2/Co3O4 was around 4.7. After irradiation in the solar box, more than 98% of the Reactive Black 5 was photocatalytically degraded within 60 min.