Intine Formation Research Articles

Temperature and light reverse the fertility of rice P/TGMS line ostms19 via reactive oxygen species homeostasis.

P/TGMS (Photo/thermo-sensitive genic male sterile) lines are crucial resources for two-line hybrid rice breeding. Previous studies revealed that slow development is a general mechanism for sterility-fertility conversion of P/TGMS in Arabidopsis. However, the difference in P/TGMS genes between rice and Arabidopsis suggests the presence of a distinct P/TGMS mechanism in rice. In this study, we isolated a novel P/TGMS line, ostms19, which shows sterility under high-temperature conditions and fertility under low-temperature conditions. OsTMS19 encodes a novel pentatricopeptide repeat (PPR) protein essential for pollen formation, in which a point mutation GTA(Val) to GCA(Ala) leads to ostms19 P/TGMS phenotype. It is highly expressed in the tapetum and localized to mitochondria. Under high temperature or long-day photoperiod conditions, excessive ROS accumulation in ostms19 anthers during pollen mitosis disrupts gene expression and intine formation, causing male sterility. Conversely, under low temperature or short-day photoperiod conditions, ROS can be effectively scavenged in anthers, resulting in fertility restoration. This indicates that ROS homeostasis is critical for fertility conversion. This relationship between ROS homeostasis and fertility conversion has also been observed in other tested rice P/TGMS lines. Therefore, we propose that ROS homeostasis is a general mechanism for the sterility-fertility conversion of rice P/TGMS lines.

Full-length EFOP3 and EFOP4 proteins are essential for pollen intine development in Arabidopsis thaliana.

Pollen development is critical to plant reproduction, but the underlying regulatory molecular mechanisms have not been fully elucidated. The Arabidopsis (Arabidopsis thaliana) EFR3 OF PLANT 3 (EFOP3) and EFR3 OF PLANT 4 (EFOP4) genes encode members of the Armadillo (ARM) repeat superfamily that play key roles in pollen development. Herein, we demonstrate that EFOP3 and EFOP4 are co-expressed in pollen at anther stages 10-12, but loss-of-function of both EFOP3 and EFOP4 leads to male gametophyte sterility, irregular intine, and shriveled pollen grains at anther stage 12. We further established that full-length EFOP3 and EFOP4 specifically localize to the plasma membrane, and the integrity of these proteins is essential for pollen development. We observed uneven intine, less organized cellulose and reduced pectin content in mutant pollen compared with the wild-type. These, together with the misexpression of several genes related to cell wall metabolism in efop3-/- efop4+/- mutants, suggest that EFOP3 and EFOP4 may indirectly regulate the expression of these genes to affect intine formation, thus controlling Arabidopsis pollen fertility in a functionally redundant manner. Moreover, transcriptome analysis showed that the absence of EFOP3 and EFOP4 function affects multiple pollen development pathways. These results enhance our understanding of EFOPs proteins and their role in pollen development.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Arabidopsis Novel Microgametophyte Defective Mutant 1 Is Required for Pollen Viability via Influencing Intine Development in Arabidopsis.

The pollen intine layer is necessary for male fertility in flowering plants. However, the mechanisms behind the developmental regulation of intine formation still remain largely unknown. Here, we identified a positive regulator, Arabidopsis novel microgametophyte defective mutant 1 (AtNMDM1), which influences male fertility by regulating intine formation. The AtNMDM1, encoding a pollen nuclei-localized protein, was highly expressed in the pollens at the late anther stages, 10–12. Both the mutations and the knock-down of AtNMDM1 resulted in pollen defects and significantly lowered the seed-setting rates. Genetic transmission analysis indicated that AtNMDM1 is a microgametophyte lethal gene. Calcofluor white staining revealed that abnormal cellulose distribution was present in the aborted pollen. Ultrastructural analyses showed that the abnormal intine rather than the exine led to pollen abortion. We further found, using transcriptome analysis, that cell wall modification was the most highly enriched gene ontology (GO) term used in the category of biological processes. Notably, two categories of genes, Arabinogalactan proteins (AGPs) and pectin methylesterases (PMEs) were greatly reduced, which were associated with pollen intine formation. In addition, we also identified another regulator, AtNMDM2, which interacted with AtNMDM1 in the pollen nuclei. Taken together, we identified a novel regulator, AtNMDM1 that affected cellulose distribution in the intine by regulating intine-related gene expression; furthermore, these results provide insights into the molecular mechanisms of pollen intine development.

Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development.

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106×G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

Pollen development in three selected species of Rubiaceae provides ontogenetic evidence for pollen evolution

Pollen development in Ixora chinensis, Pentas lanceolata, and Spermacoce pusilla was investigated. Simultaneous cytokinesis at meiosis, a persistent epidermis, fibrous thickenings in the endothecium, an ephemeral middle layer, and a secretory tapetum that remains uninucleate were observed in all examined species and constitute a family character in Rubiaceae. The main ontogenetic sequence of sporoderm development is generally centripetal and shows major similarities among the studied species, with slight differences in the timing of specific events. Tapetum degradation starts slightly later in I. chinensis than in P. lanceolata and S. pusilla, while intine formation may be slightly later in S. pusilla than in the other two species. Occurrences of orbicules, white-line-centered endexine lamellae and apertural intine projections were observed in I. chinensis and P. lanceolata but not in S. pusilla. Columellae are well-developed in I. chinensis and P. lanceolata but are simplified in S. pusilla. Carbohydrate reserves in mature pollen grains also vary among these three species. Thickened endexine at the apertural sites and a membranous granular layer are shared by the three species. White-line-centered lamellae might represent a common mechanism for endexine formation in the family. Endexine dilations and thickening occurred in the apertural region represent a common character in the family. The ephemeral membranous granular layer developed beneath the endexine is a very common feature for Rubiaceae. Our study shows that ultrastructural and developmental studies are essential in illustrating micromorphological features of pollen especially these ephemeral characters. Thus, application of palynological ontogenetic data to phylogenetic problems deserves more attention.

Slowing Development Facilitates Arabidopsis mgt Mutants to Accumulate Enough Magnesium for Pollen Formation and Fertility Restoration.

Magnesium (Mg) is an abundant and important cation in cells. Plants rely on Mg transporters to take up Mg from the soil, and then Mg is transported to anthers and other organs. Here, we showed that MGT6+/− plants display reduced fertility, while mgt6 plants are fertile. MGT6 is expressed in the anther at the early stages. Pollen mitosis and intine formation are impaired in aborted pollen grains (PGs) of MGT6+/− plants, which is similar to the defective pollen observed in mgt5 and mgt9 mutants. These results suggest that Mg deficiency leads to pollen abortion in MGT6+/− plants. Our data showed that mgt6 organs including buds develop significantly slower and mgt6 stamens accumulate a higher level of Mg, compared with wild-type (WT) and MGT6+/− plants. These results indicate that slower bud development allows mgt6 to accumulate sufficient amounts of Mg in the pollen, explaining why mgt6 is fertile. Furthermore, we found that mgt6 can restore fertility of mgt5, which has been reported to be male sterile due to defects in Mg transport from the tapetum to microspores and that an additional Mg supply can restore its fertility. Interestingly, mgt5 fertility is recovered when grown under short photoperiod conditions, which is a well-known factor regulating plant fertility. Taken together, these results demonstrate that slow development is a general mechanism to restore mgts fertility, which allows other redundant magnesium transporter (MGT) members to transport sufficient Mg for pollen formation.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Arabidopsis Cys2/His2 zinc-finger protein MAZ1 is essential for intine formation and exine pattern

Cys2/His2 zinc-finger protein (C2H2-ZFP) is widely involved in the reproductive development of plants, but its role in pollen development is still elusive. Here, we identified a pollen-related C2H2-ZFP gene named as MALE FERTILITY-ASSOCIATED ZINC FINGER PROTEIN 1 (MAZ1), which was first isolated from Arabidopsis thaliana. MAZ1 showed a preferential expression pattern in early anther development. Its mutation resulted in aberrant primexine deposition at the tetrad stage, followed by a defective multiple-layer pattern of exine with irregular baculum and no tectum. Furthermore, microspore development was arrested, and no intine layer was formed. These developmental defects led to fertility reduction and pollen abortion. This study reveals the essential role of MAZ1 in pollen wall development.

BcPME37c is involved in pollen intine formation in Brassica campestris

Pollen wall development is one of the key processes of pollen development. Several pectin methylesterase (PME) genes participate in pollen germination and pollen tube growth. However, the relationship between PME genes and pollen intine formation remains unclear. In this study, we investigated the expression and subcellular localization of the PME gene BcPME37c in Brassica campestris. Furthermore, morphology and cytology methods were used to examine the phenotype of the CRISPR/Cas9 system-induced BcPME37c mutant. We found that BcPME37c is predominately expressed in mature stamen and located at the cell wall. BcPME37c mutation causes the abnormal thickening of the pollen intine of B. campestris. Our study indicated that BcPME37c is required for pollen intine formation in B. campestris.

Constitutive overexpression of the classical arabinogalactan protein gene BcMF18 in Arabidopsis causes defects in pollen intine morphogenesis

Arabinogalactan proteins (AGPs) are essential members of hyperglycosylated glycoproteins and are critical for the sexual reproduction of plants. To our knowledge, currently Arabodopsis FLA3 and Brassica BcMF8 are the only two members of AGPs reported concerning pollen intine formation. Previously, the orthologue of AtAGP6 in B. campestris, BcMF18, was isolated and proved to affect microspore development. In this study, western blot assay and subcellular localization showed that BcMF18 encoded an approximately 19.5 kDa secretory AGP protein which anchored to extracellular matrices. Ectopic overexpression of BcMF18 (OE) in Arabodopsis caused reduced male fertility and resulted in short siliques with low seed set. Approximately 46% of OE pollen grains were aborted and lacked viability, without all cytoplasmic materials and nuclei. Pollen abortion which led to defective microspore production with no cellulose was observed to start at the bicellular stage. At this point, intine layer displayed abnormalities and degraded from the bicellular stage, which led to the failure in keeping the internal environment stability and resulted in hollow remnants of microspores. However, the basic structure and patterning of the exine layer of collapsed pollen grains seemed unaffected, completed with intact nexine, baculum, and tectum to constitute a pronounced reticulate structure filled with typhine. Based on these results presented here, it is proposed that BcMF18 functions in Arabodopsis and is associated with pollen intine formation, possibly through participating in the formation of proteoglycan structures, which provide strong evidence for subfunctionalization from AtAGP6 to BcMF18.

The putative pectin methylesterase gene, BcMF23a, is required for microspore development and pollen tube growth in Brassica campestris.

BcMF23a contributes to pollen wall development via influencing intine construction, which, in turn, influences pollen tube growth. Pollen wall, the morphological out face of pollen, surrounds male gametophyte and plays an important role in plant reproduction. Pectin methylesterases (PMEs) are involved in pollen wall construction by de-esterifying pectin of the intine. In this study, the function of a putative pectin methylesterase gene, Brassica campestris Male Fertility 23a (BcMF23a), was investigated. Knockdown of BcMF23a by artificial microRNA (amiRNA) technology resulted in abnormal pollen intine formation outside of the germinal furrows at the binucleate stage. At the trinucleate stage, 20.69% of pollen possessed the degradation of nuclei, cytoplasm and the intine, resulting in shrunken pollen, whereas the remaining 75.86% were wall-disrupted with degrading cytoplasm and broken exine inside the germinal furrows. In addition, pollen abortion in transgenic plants caused germination percentage reduction by 19% in vitro and pollen tube growth disruption in natural stigma in vivo. Taken together, BcMF23a is involved in pollen development and pollen tube growth, possibly via participating in intine construction. This study may contribute towards understanding the function of pollen-specific PMEs and the molecular regulatory network of pollen wall development.

It is a matter of timing: asynchrony during pollen development and its consequences on pollen performance in angiosperms-a review.

Functional pollen is needed to successfully complete fertilization. Pollen is formed inside the anthers following a specific sequence of developmental stages, from microsporocyte meiosis to pollen release, that concerns microsporocytes/microspores and anther wall tissues. The processes involved may not be synchronous within a flower, an anther, and even a microsporangium. Asynchrony has been barely analyzed, and its biological consequences have not been yet assessed. In this review, different processes of pollen development and lifetime, stressing on the possible consequences of their differential timing on pollen performance, are summarized. Development is usually synchronized until microsporocyte meiosis I (occasionally until meiosis II). Afterwards, a period of mostly asynchronous events extends up to anther opening as regards: (1) meiosis II (sometimes); (2) microspore vacuolization and later reduction of vacuoles; (3) amylogenesis, amylolysis, and carbohydrate inter-conversion; (4) the first haploid mitosis; and (5) intine formation. Asynchrony would promote metabolic differences among developing microspores and therefore physiologically heterogeneous pollen grains within a single microsporangium. Asynchrony would increase the effect of competition for resources during development and pollen tube growth and also for water during (re)hydration on the stigma. The differences generated by developmental asynchronies may have an adaptive role since more efficient pollen grains would be selected with regard to homeostasis, desiccation tolerance, resilience, speed of (re)hydration, and germination. The performance of each pollen grain which landed onto the stigma will be the result of a series of selective steps determined by its development, physiological state at maturity, and successive environmental constrains.

Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis.

During anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP-MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5-His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum-specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high-Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.

PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation

Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation.

Cytochemical investigation at different microsporogenesis phases of male sterility in wheat, as induced by the chemical hybridising agent SQ-1

This study used semi-thin sectioning and cytochemistry to investigate the relationship between pollen nutrient metabolism and pollen abortion in male sterile lines of wheat induced by SQ-1 (a chemical hybridising agent). Anthers were collected from the tetrad to trinucleate stages, and 4ʹ,6-diamidino-2-phenylindole staining was used to visualise nuclei and confirm the development stage. Sudan Black B, periodic acid–Schiff, Coomassie Brilliant Blue, and toluidine blue were used to detect lipids, starch, proteins, and acidic polyanions, respectively. Semi-thin sectioning indicated that nutrient accumulation was much higher in the fertile line 1376 than in the sterile line 1376-PHYMS. Further, no lipids were found in the free microspore stage in the sterile line; however, at the late microspore stage, more proteins and acidic polyanions were found in the sterile line 1376-PHYMS pollen than in the fertile line 1376 pollen. From the binucleate to trinucleate pollen stages, the starch content was low and the intine considerably thinner in the pollen of the 1376-PHYMS line. SQ-1 probably hampered nutrient metabolism in the anthers, leading to decreased nutrient supply and abnormal intine formation, ultimately resulting in pollen abortion. A new mechanism for nutrient absorption, i.e. endocytosis of Ubisch bodies or orbicules by the intine through the germinal aperture, was revealed.

Pollen wall structure in Anigozanthos viridis (Haemodoraceae)

The exine of <em>Anigozanthos viridis</em> Endl. pollen is divided into two zones by a plane which has been referred to as a"commissural line"; in our results we refer to it as a junction plane - a more apt description. Following young stages there is a pronounced reduction in thickness of the inner exine, probably due to stretching of the exine without the addition of new material as development progresses. We describe the fibrillar zone under the exine in microspores as oncus-like. Since this onciform zone precedes microspore mitosis and intine formation the fibrillar zone is divided into two layers in mature pollen an onciform part and the intine proper - it is not a two layered intine.

Sporoderm development in Swida alba (Cornaceae), interpreted as a self-assembling colloidal system

The developmental events in microspores and the tapetum in Swida alba have been traced in detail. The first elements of the glycoprotein glycocalyx appear as hemispherical, elongated, and conical units, rooted in surrounding callose layer. These units are evidently transitional forms of spherical micelles. At the middle tetrad stage, these units (future columellae) acquire a cylindrical form and consist of clusters of radially oriented subunits (clusters of cylindrical micelles), twined round with a spiral element. At the late tetrad stage, procolumellae increase, and the tectum with microchannels is initiated at the interface of the callose and the glycocalyx layers. Cone-like ‘skeletons’ – typical crystal-like structures – of the future spines emerge on the tops of the columellae, and the primordial white-lined lamella appears as a demarcation line between the future foot layer and endexine. During disintegration of the tetrads, several new white-lined lamellae arise that are evidently formed on the base of laminate micelles with their typical gaps seen in transmission electron microscopy as white lines. After addition of the second granular layer of endexine, all the elements of exine become thick as a result of sporopollenin accumulation. The completion of sporoderm development is carried out after microspore mitosis by intine formation.