Regulation Of Pollen Tube Growth Research Articles

Transcriptomic Analysis of Self-Incompatibility in Alfalfa

Alfalfa (Medicago sativa L.) is an important forage crop worldwide, but molecular genetics and breeding research in this species are hindered by its self-incompatibility (SI). Although the mechanisms underlying SI have been extensively studied in other plant families, SI in legumes, including alfalfa, remains poorly understood. Here, we determined that self-pollinated pollen tubes could germinate on the stigma of alfalfa, grow through the style, and reach the ovarian cavity, but the ovules collapsed ~48 h after self-pollination. A transcriptomic analysis of dissected pistils 24 h after self-pollination identified 941 differently expressed genes (DEGs), including 784 upregulated and 157 downregulated genes. A gene ontology (GO) analysis showed that the DEGs were highly enriched in functions associated with the regulation of pollen tube growth and pollen germination. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that pentose and glucuronate interconversion, plant hormone signal transduction, the spliceosome, and ribosomes might play important roles in SI. Our co-expression analysis showed that F-box proteins, serine/threonine protein kinases, calcium-dependent protein kinases (CDPKs), bHLHs, bZIPs, and MYB-related family proteins were likely involved in the SI response. Our study provides a catalog of candidate genes for further study to understand SI in alfalfa and related legumes.

PbrBZR1 interacts with PbrARI2.3 to mediate brassinosteroid-regulated pollen tube growth during self-incompatibility signaling in pear.

S-RNase-mediated self-incompatibility (SI) prevents self-fertilization and promotes outbreeding to ensure genetic diversity in many flowering plants, including pear (Pyrus sp.). Brassinosteroids (BRs) have well-documented functions in cell elongation, but their molecular mechanisms in pollen tube growth, especially in the SI response, remain elusive. Here, exogenously applied brassinolide (BL), an active BR, countered incompatible pollen tube growth inhibition during the SI response in pear. Antisense repression of BRASSINAZOLE-RESISTANT1 (PbrBZR1), a critical component of BR signaling, blocked the positive effect of BL on pollen tube elongation. Further analyses revealed that PbrBZR1 binds to the promoter of EXPANSIN-LIKE A3 (PbrEXLA3) to activate its expression. PbrEXLA3 encodes an expansin that promotes pollen tube elongation in pear. The stability of dephosphorylated PbrBZR1 was substantially reduced in incompatible pollen tubes, where it is targeted by ARIADNE2.3 (PbrARI2.3), an E3 ubiquitin ligase that is strongly expressed in pollen. Our results show that during the SI response, PbrARI2.3 accumulates and negatively regulates pollen tube growth by accelerating the degradation of PbrBZR1 via the 26S proteasome pathway. Together, our results show that an ubiquitin-mediated modification participates in BR signaling in pollen and reveal the molecular mechanism by which BRs regulate S-RNase-based SI.

ALA3 regulates pollen tube growth and guidance by keeping PRKs in all the right places.

ALA3 regulates pollen tube growth and guidance by keeping PRKs in all the right places.

The phospholipid flippase ALA3 regulates pollen tube growth and guidance in Arabidopsis

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.

Cellulose accumulation mediated by PbrCSLD5, a cellulose synthase-like protein, results in cessation of pollen tube growth in Pyrus bretschneideri.

Cellulose, a key component of the cell wall, plays an important role in maintaining the growth of pollen tubes. However, the molecular mechanism of cellulose participating in the cessation of pear pollen tube growth remains unclear. Here, we reported that at 15 h post-cultured (HPC), the slow-growth pear pollen tubes showed thickened cell walls and cellulose accumulation in the inner wall. Transcriptome data and quantitative real-time PCR analysis showed that PbrCSLD5, a cellulose synthesis-like gene, was highly expressed in the 15 HPC pear pollen tubes. Knockdown of PbrCSLD5 caused a decrease in cellulose content in pear pollen tubes. Moreover, PbrCSLD5 overexpression in Arabidopsis resulted in the accumulation of cellulose and disruption of normal pollen tube growth. Transcription factor PbrMADS52 was found to bind to the promoter of PbrCSLD5 and enhanced its expression. Our results suggested that the PbrMADS52-PbrCSLD5 signaling pathway led to increased cellulose content in the pear pollen tube cell wall, thereby inhibiting pollen tube growth. These results provided new insights into the regulation of pollen tube growth.

Calmodulin Domain Protein Kinase PiCDPK1 Regulates Pollen Tube Growth Polarity through Interaction with RhoGDI.

The pollen-specific calcium-dependent protein kinase PiCDPK1 of Petunia inflata has previously been shown to regulate polarity in tip growth in pollen tubes. Here we report the identification of a Rho Guanine Dissociation Inhibitor (PiRhoGDI1) as a PiCDPK1 interacting protein. We demonstrate that PiRhoGDI1 and PiCDPK1 interact in a yeast 2-hybrid assay, as well as in an in vitro pull-down assay, and that PiRhoGDI1 is phosphorylated by PiCDPK1 in vitro. We further demonstrate the PiRhoGDI1 is capable of rescuing the loss of growth polarity phenotype caused by over-expressing PiCDPK1 in vivo using stable transgenic plants. We confirmed that PiRhoGDI1 interacts with a pollen-expressed ROP GTPase isoform consistent with the established role of RhoGDIs in negatively regulating GTPases through their membrane removal and locking them in an inactive cytosolic complex. ROP is a central regulator of polarity in tip growth, upstream of Ca2+, and PiCDPK1 over-expression has been previously reported to lead to dramatic elevation of cytosolic Ca2+ through a positive feedback loop. The discovery that PiCDPK1 impacts ROP regulation via PiRhoGDI1 suggests that PiCDPK1 acts as RhoGDI displacement factor and leads us to propose a model which we hypothesize regulates the rapid recycling of ROP GTPase at the pollen tube tip.

A small Rho GTPase OsRacB is required for pollen germination in rice

Plant Rho small GTPases (Rop/Rac) are versatile molecular switches regulating many plant developmental processes. Particularly, their important functions in regulating pollen development have been demonstrated in Arabidopsis. A group of conserved Rop/Rac activators RopGEFs were recently reported to regulate rice (Oryza sativa) pollen tube germination, indicating that rice and Arabidopsis may have a conserved Rop/Rac mediated signaling pathway in regulating pollen tube growth. However, the Rop/Rac activated by the rice pollen specific RopGEFs remains to be identified. Here we demonstrated a Rop/Rac gene, OsRacB, co‐expressed with the mature pollen expressed OsRopGEF2/3/6/8. The knockout mutants were normal in anther and pollen development but defective in the pollen grain germination, suggesting a specific and non‐redundant role of OsRacB in the mature pollen. We further demonstrated that OsRacB is directly activated by the pollen specific expressing OsRopGEFs in vitro. Together with the previous study, we establish a RopGEF‐Rop/Rac regulon which plays essential roles in rice pollen grain germination. Our data encourage further identification of the upstream and downstream players of RopGEF‐Rop/Rac signaling in pollen germination and have agricultural implications for breeding robust seed yielding cultivars.

PbrRALF2-elicited reactive oxygen species signaling is mediated by the PbrCrRLK1L13-PbrMPK18 module in pear pollen tubes

Rapid alkalinization factors (RALFs) are cysteine-rich peptides that play important roles in a variety of biological processes, such as cell elongation and immune signaling. Recent studies in Arabidopsis have shown that RALFs regulate pollen tube growth via plasma membrane receptor-like kinases (RLKs). However, the downstream signal transduction mechanisms of RLKs in pollen tubes are unknown. Here, we identified PbrRALF2, a pear (Pyrus bretschneideri) pollen RALF peptide that inhibits pollen tube growth. We found that PbrRALF2 interacts with a malectin-like domain-containing RLK, PbrCrRLK1L13. The relative affinity between PbrRALF2 and PbrCrRLK1L13 was at the submicromolar level, which is consistent with the values of ligand–receptor kinase pairs and the physiological concentration for PbrRALF2-mediated inhibition of pollen tube growth. After binding to its extracellular domain, PbrRALF2 activated the phosphorylation of PbrCrRLK1L13 in a dose-dependent manner. We further showed that the MAP kinase PbrMPK18 is a downstream target of PbrCrRLK1L13 that mediates PbrRALF2-elicited reactive oxygen species (ROS) production. The excessive accumulation of ROS inhibits pollen tube growth. We show that MPK acts as a mediator for CrRLK1L to stimulate ROS production, which might represent a general mechanism by which RALF and CrRLK1L function in signaling pathways.

What are the key mechanisms that alter the morphology of stigmatic papillae in Arabidopsis thaliana?

ABSTRACT Pollination is one of the critical processes that determines crop yield and quality. Thus, it is an urgent need to elucidate the mechanisms underlying pollination. Our previous research has revealed a novel phenomenon that pollen attachment to stigma caused stigma shrinkage, whereas failure of pollen attachment to stigma due to the environmental stress induced elongation of stigmatic papillae. However, little is known about the mechanisms of these morphological alterations in stigmatic papillae. Since the RLK-ROPGEF-ROP network is a common mechanism for the elongation of pollen tubes and root hairs, this network may be also involved in the elongation of papillae in the stigma. In this review, we will discuss the known mechanisms regulating pollen tube growth and root hair elongation and attempt to propose an elongation mechanism of stigmatic papillae. In addition, we will suggest that the degradation of F-actin by a significant increase in Ca2+ induced by the components of pollen coat might be a putative molecular mechanism of stigmatic papillae shrinkage during pollen adhesion.

The emerging role of GABA as a transport regulator and physiological signal.

While the proposal that γ-aminobutyric acid (GABA) acts a signal in plants is decades old, a signaling mode of action for plant GABA has been unveiled only relatively recently. Here, we review the recent research that demonstrates how GABA regulates anion transport through aluminum-activated malate transporters (ALMTs) and speculation that GABA also targets other proteins. The ALMT family of anion channels modulates multiple physiological processes in plants, with many members still to be characterized, opening up the possibility that GABA has broad regulatory roles in plants. We focus on the role of GABA in regulating pollen tube growth and stomatal pore aperture, and we speculate on its role in long-distance signaling and how it might be involved in cross talk with hormonal signals. We show that in barley (Hordeum vulgare), guard cell opening is regulated by GABA, as it is in Arabidopsis (Arabidopsis thaliana), to regulate water use efficiency, which impacts drought tolerance. We also discuss the links between glutamate and GABA in generating signals in plants, particularly related to pollen tube growth, wounding, and long-distance electrical signaling, and explore potential interactions of GABA signals with hormones, such as abscisic acid, jasmonic acid, and ethylene. We conclude by postulating that GABA encodes a signal that links plant primary metabolism to physiological status to fine tune plant responses to the environment.

Competition between anthocyanin and kaempferol glycosides biosynthesis affects pollen tube growth and seed set of Malus

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits of Malus. In Malus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flower Malus genotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set.

Ethylene signaling modulates tomato pollen tube growth through modifications of cell wall remodeling and calcium gradient.

Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.

NETWORKED2-subfamily proteins regulate the cortical actin cytoskeleton of growing pollen tubes and polarised pollen tube growth.

We have recently characterised NET2A as a pollen-specific actin-binding protein that binds F-actin at the plasma membrane of growing pollen tubes. However, the role of NET2 proteins in pollen development and fertilisation have yet to be elucidated. To further characterise the role of Arabidopsis NET2 proteins in pollen development and fertilisation, we analysed the subcellular localisation of NET2A over the course of pollen grain development and investigated the role of the NET2 family using net2 loss-of-function mutants. We observed NET2A to localise to the F-actin cytoskeleton in developing pollen grains as it underwent striking structural reorganisations at specific stages of development and during germination and pollen tube growth. Furthermore, net2 loss-of-function mutants exhibited striking morphological defects in the early stages of pollen tube growth, arising from frequent changes to pollen tube growth trajectory. We observed defects in the cortical actin cytoskeleton and actin-driven subcellular processes in net2 mutant pollen tubes. We demonstrate that NET2 proteins are essential for normal actin-driven pollen development highlighting an important role for the NET2 family members in regulating pollen tube growth during fertilisation.

Characterization of the pectin methylesterase inhibitor gene family in Rosaceae and role of PbrPMEI23/39/41 in methylesterified pectin distribution in pear pollen tube.

Pectin methylesterase inhibitor gene family in the seven Rosaceae species (including three pear cultivars) is characterized and three pectin methylesterase inhibitor genes are identified to regulate pollen tube growth in pear. Pectin methylesterase inhibitor (PMEI) participates in a variety of biological processes in plants. However, the information and function of PMEI genes in Rosaceae are largely unknown. In this study, a total of 423 PMEI genes are identified in the genomes of seven Rosaceae species. The PMEI genes in pear are categorized into five subfamilies based on structural analysis and evolutionary analysis. WGD and TD are the main duplication events in the PMEI gene family of pear. Quantitative real-time PCR analysis indicates that PbrPMEI23, PbrPMEI39, and PbrPMEI41 are increasingly expressed during pear pollen tube growth. Under the treatment of recombinant proteins PbrPMEI23, PbrPMEI39 or PbrPMEI41, the content of methylesterified pectin at the region 5-20μm from the pollen tube tip significantly increases, and the growth of pear pollen tubes is promoted. These results indicate that PMEI regulates the growth of pollen tubes by changing the distribution of methylesterified pectin in the apex.

The Role of Calcium/Calcium-Dependent Protein Kinases Signal Pathway in Pollen Tube Growth.

Pollen tube (PT) growth as a key step for successful fertilization is essential for angiosperm survival and especially vital for grain yield in cereals. The process of PT growth is regulated by many complex and delicate signaling pathways. Among them, the calcium/calcium-dependent protein kinases (Ca2+/CPKs) signal pathway has become one research focus, as Ca2+ ion is a well-known essential signal molecule for PT growth, which can be instantly sensed and transduced by CPKs to control myriad biological processes. In this review, we summarize the recent progress in understanding the Ca2+/CPKs signal pathway governing PT growth. We also discuss how this pathway regulates PT growth and how reactive oxygen species (ROS) and cyclic nucleotide are integrated by Ca2+ signaling networks.

Identification of pollen and pistil polygalacturonases in Nicotiana tabacum and their function in interspecific stigma compatibility

The pollen and pistil polygalacturonases in Nicotiana tabacum were identified and found to regulate pollen tube growth and interspecific compatibility. Polygalacturonase (PG) is one of the enzymes catalyzing the hydrolysis of pectin. This process plays important roles in the pollen and pistil. In this research, the pollen and pistil PGs in Nicotiana tabacum (NtPGs) were identified, and their expression, localization and the potential function in the pollen and interspecific stigma incompatibility were explored. The results showed that 118 NtPGs were retrieved from the genome of N. tabacum. The phylogenetic tree and RT-qPCR analysis led to the identification of 10 pollen PGs; among them, two, seven and one showed specifically higher expression levels in the early development of anthers, during pollen maturation and in mature anthers, respectively, indicating their function difference. Immunofluorescence analysis showed that PGs were located in the cytoplasm of (1) mature pollen and (2) in vitro grown pollen tubes, as well as in the wall of in vivo grown pollen tubes. Four NtPGs in clade A were identified as the pistil PGs, and the pistil PGs were not found in clade E. Significantly higher PGs expression was recorded after incompatible pollination in comparison with the compatible stigma, indicating a potential function of PGs in regulating stigma incompatibility. The influence of PGs on pollen tube growth was explored in vitro and partly in vivo, showing that high PGs activity inhibited pollen tube growth. The application of PGs on the otherwise compatible stigma resulted in pollen tube growth inhibition or failure of germination. These results further supported that increased PGs expression in incompatible stigma might be partially responsible for the interspecific stigma incompatibility in Nicotiana.

Keeping up with the RALFs: how these small peptides control pollen-pistil interactions in Arabidopsis.

The pollen and pistil RALF peptides, along with multiple receptor-like kinases and leucine-rich repeat extensins, regulate pollen tube growth and the final burst within the ovule, where sperm cells are released for fertilisation to occur. This review introduces some new questions that arose about the regulation of this complex process.

LORELEI-LIKE GPI-ANCHORED PROTEINS 2/3 Regulate Pollen Tube Growth as Chaperones and Coreceptors for ANXUR/BUPS Receptor Kinases in Arabidopsis

Pollen tube growth is crucial for successful fertilization. In Arabidopsis thaliana, the ANXUR (ANX)/BUPS receptor kinase complex controls and maintains pollen tube growth in response to autocrine rapid alkalinization factor 4/19 (RALF4/19) signaling; however, the molecular and cellular mechanisms underlying the ANX/BUPS-mediated regulation of pollen tube growth remain unclear. In this study, we found that pollen-specific LORELEI-like GPI-anchored proteins 2 and 3 (LLG2/3) promote pollen tube growth in vitro and in vivo. LLG2/3 interacte with ANX/BUPS in a RALF4-concentration-dependent manner, suggesting that ANX/BUPS and LLG2/3 might form a receptor–coreceptor complex for perceiving RALF peptide signals. Disruption of the ANX/BUPS-LLG2/3 interaction led to the cytoplasmic retention of ANX1/2, in either llg2/3 knockdown mutants or in anx1/2 mutants lacking the J region, which mediates the ANX/BUPS-LLG2/3 interaction. Moreover, we found that RALF4 induced the production of reactive oxygen species (ROS), which stimulate pollen tube growth and reduce pollen burst rate. ROS levels are reduced in the pollen tubes of LLG2/3 RNAi lines, and application of exogenous H2O2 could partially rescue the defective pollen tube growth of LLG2/3 RNAi lines. Taken together, our study identifies LLG2/3 as novel regulatory components of pollen tube growth, and shows that they chaperone ANX/BUPS for secretion to the apical plasma membrane of pollen tube and act as coreceptors of ANX/BUPS in the activation of ROS production for promoting pollen tube growth.

Nicotiana tabacum pollen-pistil interactions show unexpected spatial and temporal differences in pollen tube growth among genotypes.

This research revealed diverse PTG rates among intraspecific pollen-pistil interactions that showed variable dependency on the stigma and mature TT. Pollen-pistil interactions regulate pollen tube growth (PTG) rates and are determinants of fertilization and seed set. This research focuses on the diversity of intraspecific PTG rates and the spatial and temporal regulation of PTG among Nicotiana tabacum genotypes. Nonrandom mating within self-compatible species has been noted, but little is known on the mechanisms involved. To begin research on nonrandom mating, we took advantage of the model reproductive system of N. tabacum and used seventeen diverse N. tabacum genotypes in a complete pollination diallel to measure the diversity of intraspecific pollen-pistil interactions. The 289 intraspecific interactions showed surprisingly large differences in PTG rates. The interaction between specific males and females resulted in 18 specific combining abilities that were significantly different, indicating the importance of the specific genotype interaction in regulating intraspecific PTG. No single female or male genotype exerted overall control of PTG rates, as determined by a general combining ability analysis. Slow and fast pollen-pistil interactions showed spatial differences in growth rates along the style. Slower interactions had a slower initial PTG rate while fast interactions had faster consistent rates of growth indicating spatial regulation of PTG in the pistil. Removal of the stigma or the mature transmitting tissue (TT) showed the tissue-specific component of PTG regulation. Stigma removal resulted in slower or no change in PTG rate depending on the pollen and pistil genotypes. Removal of the TT, which necessitated removal of the stigma, showed no change, slower or unexpectedly, increased growth rates relative to growth rates without a stigma. These data show the diverse nature of pollen-pistil interactions in N. tabacum genotypes providing a system to further investigate the regulation of PTG.

Study of auxin regulation of pollen tube growth through calcium channels in Pyrus pyrifolia

Although several studies have shown that auxin can promote pollen tube growth, the mechanism of regulation is unclear. Here, we report our studies showing that auxin increased pollen tube germination and pollen tube growth in Pyrus pyrifolia. This promotion markedly depended on extracellular pH. At pH 6.5 or 7.5, auxin effectively promoted pollen tube growth, but this effect disappeared at pH 4.5 or 5.5. This finding is consistent with the regulation of calcium channels by auxin. Auxin can increase the H+–ATPase activity of pollen tube. When sodium vanadate was used to inhibit H+–ATPase activity, the promotion of calcium channel activity by auxin was significantly inhibited. These results suggest that auxin promotes pollen tube growth via an effect on Ca2+ channel activity in apical pollen tubes; however, this regulation is influenced by ATPase activity.