A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation

Potassium antimonate was used to locate Ca2+ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion.

Quantitative assessment of coagulation of atmospheric particles onto airborne birch pollen grains

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

The pollen wall of Ephedra foliata was examined by LM, SEM and TEM. The pollen wall consists of three layers: ectexine, foot layer and endexine. The ectexine has a solid tectum underlain by infratectal processes. The infratectal components appear granular in dried grains but there are columellae‐like rods in fresh fixed grains that were not subject to dehydration and the elevation of ridges. The Ephedra foot layer is part of the ectexine and has the same contrast. There is a white line lamellation between the foot layer and outer component of the endexine that is probably a junction plane between units of the foot layer and the endexine. The endexine consists of many cylindrical sheets that extend parallel with the long axis of the pollen grain. These sheets appear interconnected and may be branched or recurved in some sites. In pollen that had been acetolyzed the endexine was stained lighter than the ectexine, the reverse of most fresh grains. This is due to the endexine being porous and the material held in it is easily extracted by washing, acetolysis, etc. In dried and acetolized pollen grains the ridges are elevated and the lamellae of the endexine are generally appressed. While in fresh fixed pollen the ridges are flattened and the endexine shows gaps between the lamellae. The intine is thick in fresh fixed mature pollen but we have seen no indication of areas having an exceptionally thick intine that could be considered apertural. The microsporangium of Ephedra is surrounded by a peritapetal lamellation, as in angiosperms. This lamellation extends between tapetal cells as “tapetal (cell) markers.”; The orbicules of Ephedra show a lamellation like those of conifers and angiosperms where such a lemellation results from the formative process. Orbicules of Ephedra are generally large and easily removable from the anthers, therefore they could be favorable objects for study of exine chemistry, function and structure. There is great variation in pollen morphological characteristics such as size, number and shape of ridges and outline of pollen grains from an individual plant and even within a single microsporangium. This would make it difficult to determine affinities of fossil polyplicate pollen with extant species.

The development of the pollen grains is a complex and regulated process. However, failures in sporogenesis and gametogenesis are common and include cell division errors and multiple structural modifications, culminating usually in abortions or, less frequently, in formation of anomalous pollen grains. These anomalies include altered morphology and functionality and are reported in several species, but are still unexplored in Bromeliaceae, a diverse neotropical family. Thus, this work structurally describes anomalous pollen grains in anthers of Dyckia racinae L.B. Sm., a Bromeliaceae species endemic to Southern Brazil. Mature anthers were processed and analyzed using standard methods for light microscopy. In addition to structurally normal pollen grains, unicellular, geminate, voluminous and aborted pollen grains were structurally described. These morphological alterations include differences in developmental destination, pollen grains with varied sizes, fusions with cytoplasmic communications, morphological alterations in exine and intine and variations in the cytoplasmic content. These anomalies are normally related to developmental disruptions, such as altered programmed cell death in tapetum, associated with cytoplasmic male sterility, for example, which usually leading to extensive abortion of the pollen grains in the anther locules. The structural anomalies described in this study, however, occur in anthers with apparently normal development and were first described for Bromeliaceae. Our results suggest a partial male sterility for this species, an unexplored embryological aspect of pollen grains in this family. Future investigations of ultrastructure and development of the anomalous pollen grains should contribute to a better understanding of the reported anomalies.

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

Due to the COVID-19 pandemic in early 2020, large-scale industrial production has been stagnant and reduced, the urban air quality has been greatly improved. It provided an excellent opportunity to explore the effects of air pollutants on the sensitization of pollen allergen proteins in the environment. Platanus pollen grains sampled in the spring of 2019 and 2020 were used for detailed characterization and analysis. Scanning electron microscopy, Fourier transform infrared, X-ray spectroscopy (XPS), trypan blue staining, and western blot analysis were employed to characterize Platanus pollen protein released from pollen grains. Our data showed that the viability of the pollen grains in 2019 was lower compared that in 2020, and the pollen grains collected in 2019 had a higher absorption peak of protein functional groups. The XPS spectra assay result demonstrated that the binding energy of the high-resolution components had not variation on the surface of pollen grains, but relative content of nitrogen and peptide chain in the pollen grains sampled in 2019 were higher than in 2020. These results suggested that more protein in the pollen grains was released onto the surface of pollen grains. In addition, western blot assay showed that the expression of Pla a3 protein in pollen grains sampled in 2019 was significantly higher than that in 2020, revealing that air pollutants could enhance the expression of Pla a3 proteins in Platanus pollen.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10453-021-09731-6.

Increasing allergy potency of Zinnia pollen grains in polluted areas

Pollen grains of 22 taxa from 7 sections and 2 subgenera of the genus Geranium from the territory of the Altai Mountain Country and 9 other species of the genus growing outside the study area were examined with the help of scanning electron microscope. The pollen of 10 taxa was studied for the first time. The pollen grains of all studied Geranium taxa are radially symmetric, isopolar, simple, 3-(rarely 4)-groove-ore, oblate-spheroidal or round-ellipsoidal, with pole three-(rarely four-)lobed, with equator almost round. It is shown that common character for all investigated Geranium taxa is clavate sculpture of the first row of the exine. For the first time for the genus Geranium the species G. regelii with 4-lobed, 4-groove-ore pollen grains was identified. It is shown that the features of pollen grains have a value for the taxonomy of Geranium species . Descent groups in sections Geranium and Recurvata according the pollen grains features were revealed. 3 descent groups (groups of G. sylvaticum, G. albiflorum, G. pseudosibiricum) are in the section Geranium, 2 descent groups (groups of G. pratense and G. collinum) are in the section Recurvata. Studied species of the genus Geranium differ on a set of features: the length of polar axis and equatorial diameter, the sculpture of the exine, size, shape and surface of heads, ornamentation of the surface of pollen grains. The size of pollen grains has sectional value and correlate with the size of the corolla: the pollen grains of species in section Recurvata have the largest sizes; the pollen grains of species in the section Batrachioides have the smallest size. Peculiarities of the internal reticle of the exine and the ornamentation of the surface of the pollen grains have subsectional value. The surface sculpture of the clavate heads can be a feature for division of species

Anthers containing pollen grains of Barleria obtusa at various stages of development were examined to investigate pollen wall ontogeny. Whole anthers containing pollen grains were viewed with a light microscope, firstly to identify stages of pollen grain development and secondly to assess the state of the tapetum. Developing pollen grains were sectioned with a Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) in order to determine the suitability of this method for an investigation of exine ontogeny. The pollen wall structure and in particular the development of the external elaborate, reticulate sculpturing were examined. The exine was deposited on the immature pollen grains during the tetrad phase of development with the sculpturing, although somewhat condensed, already apparent at this early stage. The subsequent stages of development show the sculpturing advancing towards its final expanded structure as the grain increases in size. The tapetum was unusual in that it not only lined the anther locule but also surrounded each tetrad group without invaginating the tetrad arrangement, thus defining it as a secretory type.

The specialized multilayered pollen wall plays multiple roles to ensure normal microspore development. The major components of the pollen wall (e.g. sporopollenin and lipidic precursors) are provided from the tapetum. Material export from the endoplasmic reticulum (ER) is mediated by coat protein complex II (COPII) vesicles. The Arabidopsis thaliana genome encodes seven homologs of SEC23, a COPII component. However, the functional importance of this diversity remains elusive. Here, we analyzed knockout and knockdown lines for AtSEC23A and AtSEC23D, two of the A. thaliana SEC23 homologs, respectively. Single atsec23a and atsec23d mutant plants, despite normal fertility, showed an impaired exine pattern. Double atsec23ad mutant plants were semi-sterile and exhibited developmental defects in pollen and tapetal cells. Pollen grains of atsec23ad had defective exine and intine, and showed signs of cell degeneration. Moreover, the development of tapetal cells was altered, with structural abnormalities in organelles. AtSEC23A and AtSEC23D exhibited the characteristic localization pattern of COPII proteins and were highly expressed in the tapetum. Our work suggests that AtSEC23A and AtSEC23D may organize pollen wall development and exine patterning by regulating ER export of lipids and proteins necessary for pollen wall formation. Also, our results shed light on the functional heterogeneity of SEC23 homologs.

Perfect flowers of Maytenus obtusifolia have partial sterility of pollen grains, resulting in collapsed and developed free microspores. However, the cellular events resulting in partial male sterility have not been determined. In pistillate flowers of this species, male sterility is related to the premature programmed cell death (PCD) in tapetum and sporogenic cells. The process occurs through autophagy via macroautophagy and massive autophagy and is associated with sporophytic cytoplasmic male sterility (CMS). Here, we characterised the development of pollen grains and investigated the cellular events that result in tapetum cells and free microspores PCD in perfect flowers, using light and transmission electron microscopy combined with the TUNEL (Terminal deoxynucleotidyl transferase mediated dUDP end-Labeling) assay and the ZIO (Zinc iodide-osmium tetroxide) method. Pollen grain development in perfect flowers was divided into eight developmental stages based on the characteristics of the pollen grains. Tapetum cells undergo PCD at the free microspore stage, through a macroautophagic process, by formation of autophagosomes and by autophagosomes giving rise to lytic vacuoles at maturity. In the final stage of PCD, massive autophagy occurs by rupture of the tonoplast. The development of viable and inviable microspores diverges at the vacuolated microspore stage, when PCD occurs in some free microspores, causing interruption of pollen development through necrosis. These events result in the observed partial male sterility. Viable microspores undergo mitosis and develop into tricellular pollen grains. Male sterility in hermaphrodite individuals is here interpreted as gametophytic CMS.

Novel organelles and targeting mechanisms in the anther tapetum

PRODUCTION OF SPOROPOLLENIN BY THE TAPETUM

Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus