Ostkpr1 functions in anther cuticle development and pollen wall formation in rice.
ABSTRACT: BACKGROUND:During pollen wall formation in flowering plants, a conserved metabolon consisting of acyl-CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide ?-pyrone reductase (TKPR), is required for sporopollenin synthesis. Despite this, the precise function of each of these components in different species remains unclear. RESULTS:In this study, we characterized the function of OsTKPR1, a rice orthologue of Arabidopsis TKPR1. Loss of function of OsTKPR1 delayed tapetum degradation, reduced the levels of anther cuticular lipids, and impaired Ubisch body and pollen exine formation, resulting in complete male sterility. In addition, the phenylpropanoid pathway in mutant anthers was remarkably altered. Localization studies suggest that OsTKPR1 accumulates in the endoplasmic reticulum, while specific accumulation of OsTKPR1 mRNA in the anther tapetum and microspores is consistent with its function in anther and pollen wall development. CONCLUSIONS:Our results show that OsTKPR1 is indispensable for anther cuticle development and pollen wall formation in rice, providing new insights into the biochemical mechanisms of the conserved sporopollenin metabolon in flowering plants.
Project description:Male reproductive development involves a complex series of biological events and precise transcriptional regulation is essential for this biological process in flowering plants. Several transcriptional factors have been reported to regulate tapetum and pollen development, however the transcriptional mechanism underlying Ubisch bodies and pollen wall formation remains less understood. Here, we characterized and isolated a male sterility mutant of TDR INTERACTING PROTEIN 3 (TIP3) in rice. The tip3 mutant displayed smaller and pale yellow anthers without mature pollen grains, abnormal Ubisch body morphology, no pollen wall formation, as well as delayed tapetum degeneration. Map-based cloning demonstrated that TIP3 encodes a conserved PHD-finger protein and further study confirmed that TIP3 functioned as a transcription factor with transcriptional activation activity. TIP3 is preferentially expressed in the tapetum and microspores during anther development. Moreover, TIP3 can physically interact with TDR, which is a key component of the transcriptional cascade in regulating tapetum development and pollen wall formation. Furthermore, disruption of TIP3 changed the expression of several genes involved in tapetum development and degradation, biosynthesis and transport of lipid monomers of sporopollenin in tip3 mutant. Taken together, our results revealed an unprecedented role for TIP3 in regulating Ubisch bodies and pollen exine formation, and presents a potential tool to manipulate male fertility for hybrid rice breeding.
Project description:In flowering plants, ideal male reproductive development requires the systematic coordination of various processes, in which timely differentiation and degradation of the anther wall, especially the tapetum, is essential for both pollen formation and anther dehiscence. Here, we show that OsGPAT3, a conserved glycerol-3-phosphate acyltransferase gene, plays a critical role in regulating anther wall degradation and pollen exine formation. The gpat3-2 mutant had defective synthesis of Ubisch bodies, delayed programmed cell death (PCD) of the inner three anther layers, and abnormal degradation of micropores/pollen grains, resulting in failure of pollen maturation and complete male sterility. Complementation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) experiments demonstrated that OsGPAT3 is responsible for the male sterility phenotype. Furthermore, the expression level of tapetal PCD-related and nutrient metabolism-related genes changed significantly in the gpat3-2 anthers. Based on these genetic and cytological analyses, OsGPAT3 is proposed to coordinate the differentiation and degradation of the anther wall and pollen grains in addition to regulating lipid biosynthesis. This study provides insights for understanding the function of GPATs in regulating rice male reproductive development, and also lays a theoretical basis for hybrid rice breeding.
Project description:<h4>Background</h4>Decreased spikelet fertility is often responsible for reduction in grain yield in rice (Oryza sativa L.). In this study, two varieties with different levels of heat tolerance, Liangyoupeijiu (LYPJ, heat susceptible) and Shanyou63 (SY63, heat tolerant) were subjected to two temperature treatments for 28 days during the panicle initiation stage in temperature/relative humidity-controlled greenhouses: high temperature (HT; 37/27 °C; day/night) and control temperature (CK; 31/27 °C; day/night) to investigate changes in anther development under HT during panicle initiation and their relationship with spikelet fertility.<h4>Results</h4>HT significantly decreased the grain yield of LYPJ by decreasing the number of spikelets per panicle and seed setting percentage. In addition, HT produced minor adverse effects in SY63. The decreased spikelet fertility was primarily attributed to decreased pollen viability and anther dehiscence, as well as poor pollen shedding of the anthers of LYPJ under HT. HT resulted in abnormal anther development (fewer vacuolated microspores, un-degraded tapetum, unevenly distributed Ubisch bodies) and malformation of pollen (obscure outline of the pollen exine with a collapsed bacula, disordered tectum, and no nexine of the pollen walls, uneven sporopollenin deposition on the surface of pollen grains) in LYPJ, which may have lowered pollen viability. Additionally, HT produced a compact knitted anther cuticle structure of the epidermis, an un-degraded septum, a thickened anther wall, unevenly distributed Ubisch bodies, and inhibition of the confluent locule, and these malformed structures may be partially responsible for the decreased anther dehiscence rate and reduced pollen shedding of the anthers in LYPJ. In contrast, the anther wall and pollen development of SY63 were not substantially changed under HT.<h4>Conclusions</h4>Our results suggest that disturbed anther walls and pollen development are responsible for the reduced spikelet fertility and grain yield of the tested heat susceptible variety, and noninvasive anthers and pollen formation in response to HT were associated with improved heat tolerance.
Project description:<h4>Key message</h4>An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization. Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.
Project description:The anther cuticle and pollen wall function as physical barriers that protect genetic material from various environmental stresses. The anther cuticle is composed of wax and cutin, the pollen wall includes exine and intine, and the components of the outer exine are collectively called sporopollenin. Other than cuticle wax, cutin and sporopollenin are biopolymers compounds. The precise constituents and developmental mechanism of these biopolymeric are poorly understood. Here, we reported a complete male sterile mutant, male sterile6021, in maize. The mutant displayed a smooth anther surface and irregular pollen wall formation before anthesis, and its tapetum was degraded immaturely. Gas chromatography-mass spectrometry analysis revealed a severe reduction of lipid derivatives in the mutant anther. We cloned the gene by map based cloning. It encoded a fatty acyl carrier protein reductase that was localized in plastids. Expression analysis indicated that MS6021 was mainly expressed in the tapetum and microspore after the microspore was released from the tetrad. Functional complementation of the orthologous Arabidopsis mutant demonstrated that MS6021 is conserved between monocots and dicots and potentially even in flowering plants. MS6021 plays a conserved, essential role in the successful development of anther cuticle and pollen exine in maize.
Project description:<h4>Background and aims</h4>The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.<h4>Methods</h4>In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.<h4>Key results</h4>Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.<h4>Conclusions</h4>This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.
Project description:The structurally robust biopolymer sporopollenin is the major constituent of the exine layer of pollen wall and plays a vital role in plant reproductive success. The sporopollenin precursors are synthesized through an ancient polyketide biosynthetic pathway consisting of a series of anther-specific enzymes that are widely present in all land plant lineages. Tetraketide α-pyrone reductase 1 (TKPR1) and TKPR2 are two reductases catalyzing the final reduction of the carbonyl group of the polyketide synthase-synthesized tetraketide intermediates to hydroxylated α-pyrone compounds, important precursors of sporopollenin. In contrast to the functional conservation of many sporopollenin biosynthesis associated genes confirmed in diverse plant species, TKPR2's role has been addressed only in Arabidopsis, where it plays a minor role in sporopollenin biosynthesis. We identified in gerbera two non-anther-specific orthologues of AtTKPR2, Gerbera reductase 1 (GRED1) and GRED2. Their dramatically expanded expression pattern implies involvement in pathways outside of the sporopollenin pathway. In this study, we show that GRED1 and GRED2 are still involved in sporopollenin biosynthesis with a similar secondary role as AtTKPR2 in Arabidopsis. We further show that this secondary role does not relate to the promoter of the gene, AtTKPR2 cannot rescue pollen development in Arabidopsis even when controlled by the AtTKPR1 promoter. We also identified the gerbera orthologue of AtTKPR1, GTKPR1, and characterized its crucial role in gerbera pollen development. GTKPR1 is the predominant TKPR in gerbera pollen wall formation, in contrast to the minor roles GRED1 and GRED2. GTKPR1 is in fact an excellent target for engineering male-sterile gerbera cultivars in horticultural plant breeding.
Project description:Pollen fecundity is crucial to crop productivity and also to biodiversity in general. Pollen development is supported by the tapetum, a metabolically active sporophytic nurse layer that devotes itself to this process. The tapetum in cereals and a vast majority of other plants is of the nonamoeboid type. Unable to reach out to microspores, it secretes nutrients into the anther locule where the microspores reside and develop. Orbicules (Ubisch bodies), studied in various plants since their discovery approximately 140 years ago, are a hallmark of the secretory tapetum. Their significance to tapetal or pollen development has not been established. We have identified in wheat and rice an anther-specific single-copy gene (per haploid genome equivalent) whose suppression in rice by RNA interference nearly eliminated the seed set. The flowers in the transgenics were normal for female functions, but the pollen collapsed and became less viable. Further characterization of the gene product, named RAFTIN, in wheat has shown that it is present in pro-orbicule bodies and it is accumulated in Ubisch bodies. Furthermore, it is targeted to microspore exine. Although the carboxyl portion of RAFTINs shares short, dispersed amino acid sequences (BURP domain) in common with a variety of proteins of disparate biological contexts, the occurrence RAFTIN per se is limited to cereals; neither the Arabidopsis genome nor the vast collection of ESTs suggests any obvious dicot homologs. Furthermore, our results show that RAFTIN is essential for the late phase of pollen development in cereals.
Project description:7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus.
Project description:Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142<sup>OE</sup>plants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes.