RESUMO
Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Tubo Polínico , Isoformas de Proteínas , Vacúolos , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Vacúolos/metabolismo , Citoesqueleto de Actina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Complexo de Golgi/metabolismo , Mutação/genéticaRESUMO
Although DNA methylation primarily represses TEs, it also represses select genes that are methylated in plant body tissues but demethylated by DNA glycosylases (DNGs) in endosperm or pollen. Either one of two DNGs, MATERNAL DEREPRESSION OF R1 (MDR1) or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen-segregating mutations in both genes, we identify 58 candidate DNG target genes that account for 11.1% of the wild-type transcriptome but are silent or barely detectable in other tissues. They are unusual in their tendency to lack introns but even more so in their TE-like methylation (teM) in coding DNA. The majority have predicted functions in cell wall modification, and they likely support the rapid tip growth characteristic of pollen tubes. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with the potential for extremely high expression in pollen but constitutive silencing elsewhere.
Assuntos
DNA Glicosilases , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Pólen , Zea mays , Zea mays/genética , DNA Glicosilases/metabolismo , DNA Glicosilases/genética , Pólen/genética , Pólen/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutação , Tubo Polínico/metabolismo , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimentoRESUMO
Potassium (K+) is an essential macronutrient which contributes to osmotic- and turgor-related processes in plants. Calcineurin-B like Interacting Protein Kinases (CIPKs) play crucial roles in plants under low-K+ supply since they activate root K+ uptake transport systems such as AKT1 and AtHAK5. In Arabidopsis, AtCIPK9 is important for low-K+ tolerance since atcipk9 plants exhibited poor growth and leaf chlorosis when K+ was scarce. Part of these phenotypes could be ascribed to the activation of AtHAK5 by AtCIPK9. It has been reported that important differences exist between Arabidopsis and other plant species such as tomato with respect to the regulation of K+ uptake systems. Thus, our aim was to evaluate the contribution of SlCIPK9, the homologous protein of AtCIPK9 in tomato, to K+ nutrition. Unexpectedly, phenotyping experiments carried out with slcipk9 loss-of-function mutants revealed that SlCIPK9 did not play a clear role in tomato K+ homeostasis. By contrast, it was found that SlCIPK9 contributed to pollen tube elongation, but not to pollen germination, via a K+-independent mechanism. Therefore, our results highlight the remarkable differences that exist in Ca2+ signaling pathways between plant species and encourage the realization of more comparative studies as the one presented here.
Assuntos
Proteínas de Plantas , Tubo Polínico , Potássio , Solanum lycopersicum , Solanum lycopersicum/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Potássio/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Torreya grandis, a dioecious Taxaceae species of significant economic value in southeast China, presents challenges for natural pollination due to asynchronous maturation of its sex organs and low pollen vitality. In order to enhance fertilization success through artificial pollination of T. grandis, this study investigated the optimal conditions for in vitro pollen germination and pollen tube growth of T. grandis. The optimal in vitro growth medium was found to contain 29â¯mM sucrose, 0.8â¯mM H3BO3, 0.72â¯mM CaCl2, and 0.32â¯mM MgSO4, supplemented with 4⯵M NAA, 2⯵M GA3, and 5⯵M 2,4-D at pH=5.6. Under these conditions, we achieved a maximum pollen germination ratio of 69.99 ± 5.17â¯% and a pollen tube length of 34.38 ± 6.04⯵m after 6 days germination at 28°C. FM4-64 dye and Mitotracker Red staining revealed highly dynamics of vesicles and mitochondria during germination, which were accumulated at the tip of pollen tube and exhibited biphasic movement patterns. The total number, motion rate, and movement velocity of vesicles as well as mitochondria showed an initially increase followed by a gradual decrease pattern. The presence of sucrose in the medium significantly increased the dynamics and metabolic activity of both vesicles and mitochondria, which may relate with higher pollen germination ratio and faster pollen tube growth compared to sucrose-depleted conditions. Thus, these findings shed light on the physiological characteristics of Torreya pollen germination and provide scientific information for improving Torreya fruit yield through artificial pollination.
Assuntos
Germinação , Tubo Polínico , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/fisiologia , Tubo Polínico/metabolismo , Germinação/fisiologia , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Polinização , Sacarose/metabolismoRESUMO
Reproduction in angiosperms relies on the precise growth of pollen tubes, facilitating the delivery of sperm cells to the ovule for double fertilization. LATERAL ORGAN BOUNDARIES DOMAIN10 (LBD10), a plant-specific transcription factor, plays a pivotal role in Arabidopsis pollen development. Here, we uncovered LBD10's function in sustaining pollen tube growth and integrity. The lbd10 mutant exhibited elevated levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) in both pollen grains and tubes, leading to compromised pollen tube growth. The inhibition of ROS synthesis and scavenging of excess ROS with an antioxidant treatment each alleviated these defects in lbd10. The lbd10 mutant displayed reduced flavonol accumulation in both pollen grains and tubes. All the altered phenotypes of lbd10 were complemented by expressing LBD10 under its native promoter. Exogenous application of flavonoids recused the defects in pollen tube growth and integrity in lbd10, along with reducing the excess levels of ROS and H2O2. LBD10 directly binds the promoters of key flavonol biosynthesis genes in chromatin and promotes reporter gene expression in Arabidopsis mesophyll protoplasts. Our findings indicate that LBD10 modulates ROS homeostasis by transcriptionally activating genes crucial for flavonol biosynthesis, thereby maintaining pollen tube growth and integrity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flavonóis , Regulação da Expressão Gênica de Plantas , Homeostase , Peróxido de Hidrogênio , Tubo Polínico , Espécies Reativas de Oxigênio , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Flavonóis/metabolismo , Flavonóis/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Tubo Polínico/efeitos dos fármacos , Tubo Polínico/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Mutação/genética , Regiões Promotoras Genéticas/genética , FenótipoRESUMO
Pollen tubes are typical polarized growth cells whose elongation occurs only in tip regions and is highly dependent on precise and ordered exocytosis/endocytosis in the top regions of the tubes. Although anionic phospholipids have been proven to be involved in regulating vesicle trafficking and the proper localization and functions of proteins in pollen tubes, the underlying cellular and molecular mechanisms remain poorly understood. To further understand how anionic phospholipids are involved in vesicle trafficking and in the control of protein localization and functions, assay methods to analyze the polar localization of anionic phospholipids and their binding proteins, and identifying phospholipid-protein interactions, should be developed. Here, we describe detailed protocols for analyzing anionic phospholipid polar localization and colocalization with their binding proteins in Arabidopsis pollen tubes and examining phospholipid-protein interactions in vitro.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fosfolipídeos , Tubo Polínico , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Fosfolipídeos/metabolismo , Fosfolipídeos/análise , Proteínas de Arabidopsis/metabolismo , Ligação Proteica , Proteínas de Transporte/metabolismo , Ânions/metabolismoRESUMO
The pistil is the most important organ for fertilization in flowering plants, and the stigmatic papilla cells are responsible for pollen acceptance and pollen tube germination. Arabidopsis plants possess dry stigmas exhibiting high selectivity for compatible pollen. When compatible pollens are recognized and accepted by stigmatic papilla cells, water and nutrients are then transported from the stigma to pollen grains through the secretory pathway. Here, we present light microscopy-based methods for investigating autophagy and senescence of stigmatic papilla cells. These methods include the assessment of viability of stigmatic papilla cells using dual staining with fluorescein diacetate/propidium iodide, as well as the examination of vacuolar-accumulated proteins during stigma senescence. These methods can be used to understand the functions of the stigma tissue from a subcellular perspective.
Assuntos
Arabidopsis , Autofagia , Arabidopsis/fisiologia , Arabidopsis/citologia , Autofagia/fisiologia , Senescência Celular , Flores/crescimento & desenvolvimento , Flores/citologia , Vacúolos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismoRESUMO
Pummelo (Citrus grandis L. Osbeck) exhibits S-RNase-based self-incompatibility (SI), during which S-RNase cytotoxicity inhibits pollen tubes in an S-haplotype-specific manner. The entry of S-RNase into self-pollen tubes triggers a series of reactions. However, these reactions are still poorly understood in pummelo. In the present study, we used S-RNases as baits to screen a pummelo pollen cDNA library and characterized a myo-inositol oxygenase (CgMIOX3) that physically interacts with S-RNases. CgMIOX3 is highly expressed in pummelo pollen tubes, and its downregulation leads to a reduction in pollen tube growth. Upon entering pollen tubes, S-RNases increase the expression of CgMIOX3 and enhance its activity by directly binding to it in an S-haplotype-independent manner. CgMIOX3 improves pollen tube growth under oxidative stress through ascorbic acid (AsA) accumulation and increases the length of self-pollen tubes. Furthermore, over-expression of CgMIOX3 increases the relative length of self-pollen tubes growing in the style of petunia (Petunia hybrida). This study provides intriguing insights into the pumelo SI system, revealing a regulatory mechanism mediated by CgMIOX3 that plays an important role in the resistance of pollen tubes to S-RNase cytotoxicity.
Assuntos
Citrus , Regulação da Expressão Gênica de Plantas , Inositol Oxigenase , Proteínas de Plantas , Tubo Polínico , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Citrus/genética , Citrus/fisiologia , Citrus/efeitos dos fármacos , Inositol Oxigenase/genética , Inositol Oxigenase/metabolismo , Ribonucleases/metabolismo , Ribonucleases/genética , Autoincompatibilidade em Angiospermas/genética , Estresse Oxidativo , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologiaRESUMO
Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Rede trans-Golgi , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Rede trans-Golgi/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Tubo Polínico/genética , Zinco/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plant pollen tubes and root hairs typically polarized tip growth. It is well established that calcium ions (Ca2+) play essential roles in maintaining cell polarity and guiding cell growth orientation. Ca2+ signals are encoded by Ca2+ channels and transporters and are decoded by a variety of Ca2+-binding proteins often called Ca2+ sensors, in which calcineurin B-like protein (CBL) proteins function by interacting with and activating a group of kinases and activate CBL-interacting protein kinases (CIPKs). Some CBL-CIPK complexes, such as CBL2/3-CIPK12/19, act as crucial regulators of pollen tube growth. Whether these calcium decoding components regulate the growth of root hairs, another type of plant cell featuring Ca2+-regulated polarized growth, remains unknown. In this study, we identified CIPK13 and CIPK18 as genes specifically expressed in Arabidopsis (Arabidopsis thaliana) root hairs. The cipk13 cipk18 double mutants showed reduced root hair length and lower growth rates. The calcium oscillations at the root hair tip were attenuated in the cipk13 cipk18 mutants as compared to the wild-type plants. Through yeast 2-hybrid screens, CBL2 and CBL3 were identified as interacting with CIPK13 and CIPK18. cbl2 cbl3 displayed a shortened root hair phenotype similar to cipk13 cipk18. This genetic analysis, together with biochemical assays showing activation of CIPK13/18 by CBL2/3, supported the conclusion that CBL2/3 and CIPK13/18 may work as Ca2+-decoding modules in controlling root hair growth. Thus, the findings that CIPK12/19 and CIPK13/18 function in pollen tube and root hair growth, respectively, illustrate a molecular mechanism in which the same CBLs recruit distinct CIPKs in regulating polarized tip growth in different types of plant cells.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Raízes de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação/genética , Sinalização do Cálcio , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/metabolismoAssuntos
Arabidopsis , Fertilidade , Flores , Tubo Polínico , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Flores/crescimento & desenvolvimento , Flores/fisiologia , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Lipídeos , Metabolismo dos LipídeosRESUMO
Pollen germination and pollen tube elongation require rapid phospholipid production and remodeling in membrane systems that involve both de novo synthesis and turnover. Phosphatidic acid phosphohydrolase (PAH) and lysophosphatidylcholine acyltransferase (LPCAT) are 2 key enzymes in membrane lipid maintenance. PAH generates diacylglycerol (DAG), a necessary precursor for the de novo synthesis of phosphatidylcholine (PC), while LPCAT reacylates lysophosphatidylcholine to PC and plays an essential role in the remodeling of membrane lipids. In this study, we investigated the synthetic defects of pah and lpcat mutations in sexual reproduction of Arabidopsis (Arabidopsis thaliana) and explored the prospect of pistil lipid provision to pollen tube growth. The combined deficiencies of lpcat and pah led to decreased pollen tube growth in the pistil and reduced male transmission. Interestingly, pistils of the lipid mutant dgat1 ameliorated the male transmission deficiencies of pah lpcat pollen. In contrast, pollination with a nonspecific phospholipase C (NPC) mutant exacerbated the fertilization impairment of the pah lpcat pollen. Given the importance of DAG in lipid metabolism and its contrasting changes in the dgat1 and npc mutants, we further investigated whether DAG supplement in synthetic media could influence pollen performance. DAG was incorporated into phospholipids of germinating pollen and stimulated pollen tube growth. Our study provides evidence that pistil-derived lipids contribute to membrane lipid synthesis in pollen tube growth, a hitherto unknown role in synergistic pollen-pistil interactions.
Assuntos
Arabidopsis , Flores , Tubo Polínico , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Fertilidade/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mutação , Diglicerídeos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/genética , Polinização , Metabolismo dos Lipídeos , Pólen/crescimento & desenvolvimento , Pólen/genética , Pólen/metabolismoRESUMO
The intricate process of male gametophyte development in flowering plants is regulated by jasmonic acid (JA) signaling. JA signaling initiates with the activation of the basic helix-loop-helix transcription factor (TF), MYC2, leading to the expression of numerous JA-responsive genes during stamen development and pollen maturation. However, the regulation of JA signaling during different stages of male gametophyte development remains less understood. This study focuses on the characterization of the plant ARID-HMG DNA-BINDING PROTEIN 15 (AtHMGB15) and its role in pollen development in Arabidopsis (Arabidopsis thaliana). Phenotypic characterization of a T-DNA insertion line (athmgb15-4) revealed delayed bolting, shorter siliques, and reduced seed set in mutant plants compared to the wild type. Additionally, AtHMGB15 deletion resulted in defective pollen morphology, delayed pollen germination, aberrant pollen tube growth, and a higher percentage of nonviable pollen grains. Molecular analysis indicated the downregulation of JA biosynthesis and signaling genes in the athmgb15-4 mutant. Quantitative analysis demonstrated that JA and its derivatives were â¼10-fold lower in athmgb15-4 flowers. Exogenous application of methyl jasmonate could restore pollen morphology and germination, suggesting that the low JA content in athmgb15-4 impaired JA signaling during pollen development. Furthermore, our study revealed that AtHMGB15 physically interacts with MYC2 to form a transcription activation complex. This complex promotes the transcription of key JA signaling genes, the R2R3-MYB TFs MYB21 and MYB24, during stamen and pollen development. Collectively, our findings highlight the role of AtHMGB15 as a positive regulator of the JA pathway, controlling the spatiotemporal expression of key regulators involved in Arabidopsis stamen and pollen development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Oxilipinas , Pólen , Transdução de Sinais , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Pólen/crescimento & desenvolvimento , Pólen/genética , Pólen/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Mutação/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/metabolismo , FenótipoRESUMO
Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro3-5 motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tubo Polínico , Prolil Hidroxilases , Arabidopsis/metabolismo , Arabidopsis/genética , Hidroxilação , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Tubo Polínico/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Prolil Hidroxilases/metabolismo , Prolil Hidroxilases/genética , Parede Celular/metabolismoRESUMO
CrRLK1L subfamily members are involved in diverse growth- and development-related processes in Arabidopsis. However, the functions of their counterparts in rice are unknown. Here, OsANX expression was detected in developing inflorescences, mature pollen grains, and growing pollen tubes, and it was localized to the plasma membrane in pollen grains and tobacco epidermal cells. Homozygous osanx progeny could not be segregated from the CRISPR/Cas9-edited mutants osanx-c1+/- and osanx-c2+/-, and such progeny were segregated only occasionally from osanx-c3+/-. Further, all three alleles showed osanx male but not female gamete transmission defects, in line with premature pollen tube rupture in osanx-c3. Additionally, osanx-c3 exhibited precocious flowering, excessively branched inflorescences, and an extremely low seed setting rate of 1.4â¯%, while osanx-c2+/- and osanx-c3+/- had no obvious defects in inflorescence development or the seed setting rate compared to wild-type Nipponbare (Nip). Consistent with this, the complemented line pPS1:OsANX-GFP/osanx-c2 (PSC), in which the lack of OsANX expression was inflorescence-specific, showed slightly earlier flowering and overly-branched panicles. Multiple inflorescence meristem transition-related and inflorescence architecture-related genes were expressed at higher levels in osanx-c3 than in Nip; thus, they may partially account for the aforementioned mutant phenotypes. Our findings broaden our understanding of the biological functions of OsANX in rice.
Assuntos
Inflorescência , Oryza , Proteínas de Plantas , Tubo Polínico , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Oryza/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inflorescência/genética , Inflorescência/crescimento & desenvolvimento , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Flores/crescimento & desenvolvimento , Flores/genética , Regulação da Expressão Gênica de PlantasRESUMO
Flowering plants rely on the polarized growth of pollen tubes to deliver sperm cells (SCs) to the embryo sac for double fertilization. In pollen, the vegetative nucleus (VN) and two SCs form the male germ unit (MGU). However, the mechanism underlying directional transportation of MGU is not well understood. In this study, we provide the first full picture of the dynamic interplay among microtubules, actin filaments, and MGU during pollen germination and tube growth. Depolymerization of microtubules and inhibition of kinesin activity result in an increased velocity and magnified amplitude of VN's forward and backward movement. Pharmacological washout experiments further suggest that microtubules participate in coordinating the directional movement of MGU. In contrast, suppression of the actomyosin system leads to a reduced velocity of VN mobility but without a moving pattern change. Moreover, detailed observation shows that the direction and velocity of VN's movement are in close correlations with those of the actomyosin-driven cytoplasmic streaming surrounding VN. Therefore, we propose that while actomyosin-based cytoplasmic streaming influences on the oscillational movement of MGU, microtubules and kinesins avoid MGU drifting with the cytoplasmic streaming and act as the major regulator for fine-tuning the proper positioning and directional migration of MGU in pollen.
Assuntos
Citoesqueleto de Actina , Actomiosina , Cinesinas , Microtúbulos , Pólen , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Cinesinas/metabolismo , Pólen/metabolismo , Actomiosina/metabolismo , Tubo Polínico/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Arabidopsis/metabolismo , Corrente Citoplasmática , Germinação/fisiologiaRESUMO
The successful interaction between pollen and stigma is a critical process for plant sexual reproduction, involving a series of intricate molecular and physiological events. After self-compatible pollination, a significant reduction in reactive oxygen species (ROS) production has been observed in stigmas, which is essential for pollen grain rehydration and subsequent pollen tube growth. Several scavenging enzymes tightly regulate ROS homeostasis. However, the potential role of these ROS-scavenging enzymes in the pollen-stigma interaction in Brassica napus remains unclear. Here, we showed that the activity of ascorbate peroxidase (APX), an enzyme that plays a crucial role in the detoxification of hydrogen peroxide (H2O2), was modulated depending on the compatibility of pollination in B. napus. We then identified stigma-expressed APX1s and generated pentuple mutants of APX1s using CRISPR/Cas9 technology. After compatible pollination, the BnaAPX1 pentuple mutants accumulated higher levels of H2O2 in the stigma, while the overexpression of BnaA09.APX1 resulted in lower levels of H2O2. Furthermore, the knockout of BnaAPX1 delayed the compatible response-mediated pollen rehydration and germination, which was consistent with the effects of a specific APX inhibitor, ρ-Aminophenol, on compatible pollination. In contrast, the overexpression of BnaA09.APX1 accelerated pollen rehydration and germination after both compatible and incompatible pollinations. However, delaying and promoting pollen rehydration and germination did not affect the seed set after compatible and incompatible pollination in APX1 pentuple mutants and overexpression lines, respectively. Our results demonstrate the fundamental role of BnaAPX1 in pollen rehydration and germination by regulating ROS homeostasis during the pollen-stigma interaction in B. napus.
Assuntos
Ascorbato Peroxidases , Brassica napus , Proteínas de Plantas , Ascorbato Peroxidases/metabolismo , Ascorbato Peroxidases/genética , Brassica napus/genética , Brassica napus/fisiologia , Brassica napus/enzimologia , Brassica napus/metabolismo , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Germinação , Homeostase , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/fisiologia , Tubo Polínico/genética , Tubo Polínico/metabolismo , Polinização , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation.
Assuntos
Nicotiana , Proteínas de Plantas , Pólen , Tardígrados , Raios Ultravioleta , Antioxidantes/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Germinação/efeitos da radiação , Nicotiana/efeitos da radiação , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/efeitos da radiação , Pólen/metabolismo , Tubo Polínico/metabolismo , Tubo Polínico/efeitos da radiação , Tubo Polínico/genética , Estresse Fisiológico/efeitos da radiação , Tardígrados/genética , Tardígrados/metabolismoRESUMO
In angiosperms, the pollen tube enters the receptive synergid cell, where it ruptures to release its cytoplasm along with two sperm cells. This interaction is complex, and the exact signal transducers that trigger the bursting of pollen tubes are not well understood. In this study, we identify three homologous receptor-like cytoplasmic kinases (RLCKs) expressed in pollen tubes of Arabidopsis, Delayed Burst 1/2/3 (DEB1/2/3), which play a crucial role in this process. These genes produce proteins localized on the plasma membrane, and their knockout causes delayed pollen tube burst and entrance of additional pollen tubes into the embryo sac due to fertilization recovery. We show that DEBs interact with the Ca2+ pump ACA9, influencing the dynamics of cytoplasmic Ca2+ in pollen tubes through phosphorylation. These results highlight the importance of DEBs as key signal transducers and the critical function of the DEB-ACA9 axis in timely pollen tube burst in synergids.