Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Identification of self-incompatibility alleles in Quince (Cydonia oblonga Mill.).

The Quince (Cydonia oblonga Mill.), typically known for its self-compatibility, surprisingly presents a degree of self-incompatibility. This research focused on exploring the diversity within the self-incompatibility gene locus (S) in various C. oblonga genotypes. Through meticulous DNA sequencing, the study sought to unearth potential novel S alleles. In the process of genotyping the S gene across multiple quince genotypes, not only were the previously documented S1 and S2 alleles identified, but this investigation also uncovered two previously unrecognized alleles, termed S4 and S5. These alleles, particularly S4, emerged as the most prevalent among the tested genotypes. To corroborate the findings derived from DNA sequencing, the study employed pollen tube growth germination assays. These assays elucidated a higher pollen germination rate in the Ardabil2 genotype in contrast to Behta. Additionally, the study involved assessing pollen tube growth in both Ardabil2 and Behta through cross-pollination techniques, meticulously tracking the development of pollen tubes at various stages. Remarkably, the outcomes demonstrated that the Behta genotype possesses self-incompatibility, whereas the Ardabil2 genotype showcases a notable degree of self-compatibility. This groundbreaking discovery of new S alleles in quince not only affirms the species' self-compatibility but also sheds light on the complexities of allelic diversity and its impact on self-incompatibility. Such insights are invaluable for enhancing the yield of quince orchards through strategic breeding programs.

PbRbohH/J mediates ROS generation to regulate the growth of pollen tube in pear.

Respiratory burst oxidase homolog (Rboh) family genes play crucial functions in development and growth. However, comprehensive and systematic investigation of Rboh family members in Rosaceae and their specific functions during pear pollen development are still limited. In the study, 63 Rboh genes were identified from eight Rosaceae genomes (Malus domestica, Pyrus bretschneideri, Pyrus communis, Prunus persica, Rubus occidentalis, Fragaria vesca, Prunus mume and Prunus avium) and divided into seven main subfamilies (I-VII) according to phylogenetic and structural features. Different modes of gene duplication led to the expansion of Rboh family, with purifying selection playing a vital role in the evolution of Rboh genes. In addition, RNA sequencing and qRT-PCR results indicated that PbRbohH and PbRbohJ were specifically high-expressed in pear pollen. Subsequently, subcellular localization revealed that PbRbohH/J distributed at the plasma membrane. Furthermore, by pharmacological analysis and antisense oligodeoxynucleotide assay, PbRbohH/J were demonstrated to mediate the formation of reactive oxygen species (ROS) to manage pollen tube growth. In conclusion, our results provide useful insights into the functions, expression patterns, evolutionary history of the Rboh genes in pear and other Rosaceae species.

Comparative genomic analysis of the RabGAP gene family in seven Rosaceae species, and functional identification of PbrRabGAP10 in controlling pollen tube growth by mediating cellulose deposition in pear.

Rab GTPase-activating proteins (RabGAPs), serving as crucial signaling switches, play essential roles in several physiological processes related to plant growth and development. However, despite their importance, information regarding the RabGAP gene family and their biological functions remains unknown in the Rosaceae. In this study, we identified a total of 127 RabGAP genes in seven Rosaceae species, which were divided into five subfamilies. Our findings indicate that whole genome duplication (WGD) events or dispersed duplication events largely contributed to the expansion of RabGAP family members within Rosaceae species. Through tissue-specific expression analyses, we revealed that the PbrRabGAP genes exhibited distinct expression patterns in different pear tissues. Furthermore, by examining the expression pattern during pollen development and employing an antisense oligonucleotide approach, we demonstrated that PbrRabGAP10, located in the cytoplasm, mediates the imbalance of cellulose distribution, thus regulating pollen tube elongation. In conclusion, the present study offers an overview of the RabGAP family in Rosaceae genomes and serves as the basis for further functional studies.

Characterization of pollen tube development in distant hybridization of Chinese cork oak (Quercus variabilis L.).

MAIN CONCLUSION: This work mainly found that the stigma and style of Q. variabilis did not completely lose the specific recognition towards heterologous pollen, a fact which is different from previous studies. Quercus is the foundation species in the Northern Hemisphere, with extreme prevalence for interspecific hybridization. It is not yet entirely understood whether or how the pollen tube-female tissue interaction contributes to the "extensive hybridization" in oaks. Pollen storage conditions correlate with distant hybridization. We conducted hybridization experiments with Q. variabilis as female and Q. variabilis and Q. mongolica as male parents. And the differences in pollen tube (PT) development between intra- and distant interspecific hybridization were studied by fluorescence microscopy and scanning electron microscopy (SEM). Our results showed that -20 °C allowed pollen of both species to maintain some viability. Both Q. variabilis and Q. mongolica pollen germinated profusely on the stigmas. SEM results indicated that in the intraspecific hybridization, Q. variabilis pollen started to germinate at 6 h after pollination (hap), PTs elongated significantly at 12 hap, and entered the stigma at 24 hap. By contrast, Q. mongolica pollen germinated at 15 hap, and the PTs entered the stigma at 27 hap. By fluorescence microscopical studies it was observed that some PTs of Q. variabilis gathered at the style-joining at 96 hap, unlike the Q. mongolica which reached the style junction at 144 hap. The above results indicate that the abundant germination of heterologous pollen (HP) on the stigma and the "Feeble specificity recognition" of the stigma and transmitting tract to HP may create opportunities for the "extensive hybridization" of oaks. This work provides a sexual developmental reference for clarifying the causes of Quercus "extensive hybridization".

VAMP726 and VAMP725 regulate vesicle secretion and pollen tube growth in Arabidopsis.

KEY MESSAGE: VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex. Secretory vesicle fusion with the plasma membrane of the pollen tube tip is a key step in pollen tube growth. Membrane fusion was mediated by SNAREs. However, little is known about the composition and function of the SNARE complex during pollen tube tip growth. In this study, we constructed a double mutant vamp725 vamp726 via CRISPR‒Cas9. Fluorescence labeling combined with microscopic observation, luciferase complementation imaging, co-immunoprecipitation and GST pull-down were applied in the study. We show that double mutation of the R-SNAREs VAMP726 and VAMP725 significantly inhibits pollen tube growth in Arabidopsis and slows vesicle exocytosis at the apex of the pollen tube. GFP-VAMP726 and VAMP725-GFP localize mainly to secretory vesicles and the plasma membrane at the apex of the pollen tube. In addition, fluorescence recovery after photobleaching (FRAP) experiments showed that mCherry-VAMP726 colocalizes with Qa-SNARE SYP131 in the central region of the pollen tube apical plasma membrane. Furthermore, we found that VAMP726 and VAMP725 can interact with the SYP131. Based on these results, we suggest that VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex, and vesicle secretion may mainly occur at the central region of the pollen tube apical plasma membrane.

Pectate lyase-like lubricates the male gametophyte's path toward its mating partner.

The pollen tube is an extension of the male gametophyte in plants and mediates sexual reproduction by delivering the sperm cells to the female gametophyte. To accomplish this task, the elongating pollen tube must break through the thick wall of the pollen grain and penetrate multiple pistillar tissues. Both processes require the loosening of cell wall material-that of the pollen intine and that of the apoplast of the transmitting tract. The enzymatic toolbox for these cell wall modifying processes employed by the invading male gametophyte is elusive. We investigated the role of the pectin-digesting pectate lyase-like (PLL) by combining mutant analysis with microscopy observations, fluorescence recovery after photo-bleaching experiments, and immuno-detection. We show that in Arabidopsis (Arabidopsis thaliana), PLLs are required for intine loosening during the first steps of pollen tube germination. We provide evidence that during pollen tube elongation, PLLs are released by the pollen tube into the extracellular space, suggesting that they may be employed to soften the apoplast of the transmitting tissue. The synergistic enzymatic action of PLLs in the pollen grain, the pollen tube, and the transmitting track contribute to an effective fertilization process.

Differential sensitivity of ADF isovariants to a pH gradient promotes pollen tube growth.

The actin cytoskeleton is one of the targets of the pH gradient in tip-growing cells, but how cytosolic pH regulates the actin cytoskeleton remains largely unknown. We here demonstrate that Arabidopsis ADF7 and ADF10 function optimally at different pH levels when disassembling actin filaments. This differential pH sensitivity allows ADF7 and ADF10 to respond to the cytosolic pH gradient to regulate actin dynamics in pollen tubes. ADF7 is an unusual actin-depolymerizing factor with a low optimum pH in in vitro actin depolymerization assays. ADF7 plays a dominant role in promoting actin turnover at the pollen tube apex. ADF10 has a typically high optimum pH in in vitro assays and plays a dominant role in regulating the turnover and organization of subapical actin filaments. Thus, functional specification and cooperation of ADF isovariants with different pH sensitivities enable the coordination of the actin cytoskeleton with the cytosolic pH gradient to support pollen tube growth.

ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization.

The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars.

Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Disruption of pollen tube homogalacturonan synthesis relieves pollen tube penetration defects in the Arabidopsis O-FUCOSYLTRANSFERASE1 mutant.

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition.

Exploring Novel Polytubey Reproduction Pathways Utilizing Cumulative Genetic Tools.

In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Comprehensive genomic analysis of the Rho GTPases regulators in seven Rosaceae species revealed that PbrGDI1 controls pollen tube growth in Pyrus via mediating cellulose deposition.

The primary regulators of Rho GTPases are GTPase-activating protein (GAP), guanine nucleotide exchange factor (GEF), and GDP dissociation inhibitor (GDI), which function as signaling switches in several physiological processes involved in plant growth and development. This study compared how the Rho GTPase regulators functioned in seven Rosaceae species. Seven Rosaceae species, divided into three subgroups, had a total of 177 regulators of Rho GTPases. According to duplication analysis, the expansion of GEF, GAP, and GDI families was facilitated by whole genome duplication or a dispersed duplication event. The balance of cellulose deposition to control the growth of the pear pollen tube, as demonstrated by the expression profile and antisense oligonucleotide approach. Moreover, protein-protein interactions indicated that PbrGDI1 and PbrROP1 could directly interact, suggesting that PbrGDI1 regulated the growth of the pear pollen tube through PbrROP1 signaling downstream. These results lay the foundations for future functional characterization of the GAP, GEF, and GDI gene families in Pyrus bretschneideri.

RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

Elucidation of the GAUT gene family in eight Rosaceae species and function analysis of PbrGAUT22 in pear pollen tube growth.

MAIN CONCLUSION: The phylogenetic relationship and evolutionary history of the GAUT gene family were identified in 8 Rosaseae species. PbrGAUT22 was involved in controlling pollen tube growth by regulating the content of pectins. In plants, galacturonosyltransferases (GAUTs) were involved in homogalacturonan biosynthesis and functioned in maintaining pollen tube cell wall integrity. However, the feature and evolutionary history of the GAUT gene family in Rosaceae species and candidates in pear pollen tube growth remain unclear. Here, we identified 190 GAUT genes in 8 Rosaceae species, including Chinese white pear (Pyrus bretschneideri), European pear (Pyrus communis), apple (Malus × domestica), peach (Prunus persica), Japanese apricot (Prunus mume), sweet cherry (Prunus avium), woodland strawberry (Fragaria vesca) and black raspberry (Rubus occidentalis). Members in GAUT gene family were divided into 4 subfamilies according to the phylogenetic and structural analysis. Whole-genome duplication events and dispersed duplicates drove the expansion of the GAUT gene family. Among 23 pollen-expressed PbrGAUT genes in pear, PbrGAUT22 showed increased expression level during 1-6 h post-cultured pollen tubes. PbrGAUT22 was localized to the cytoplasm and plasma membrane. Knockdown of PbrGAUT22 expression in pollen tubes caused the decrease of pectin content and inhibited pear pollen tubes growth. Taken together, we investigated the identification and evolution of the GAUT gene family in Rosaceae species, and found that PbrGAUT22 played an essential role in the synthesis of pectin and the growth of pear pollen tubes.

Pollen tube emergence is mediated by ovary-expressed ALCATRAZ in cucumber.

Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube emergence. As yet, no female factor has been identified to positively regulate this transition process. In this study, we show that an ovary-expressed bHLH transcription factor Cucumis sativus ALCATRAZ (CsALC) functions in pollen tube emergence in cucumber. CsALC knockout mutants showed diminished pollen tube emergence, extremely reduced entry into ovules, and a 95% reduction in female fertility. Further examination showed two rapid alkalinization factors CsRALF4 and CsRALF19 were less expressed in Csalc ovaries compared to WT. Besides the loss of male fertility derived from precocious pollen tube rupture as in Arabidopsis, Csralf4 Csralf19 double mutants exhibited a 60% decrease in female fertility due to reduced pollen tube distribution and decreased ovule targeting efficiency. In brief, CsALC regulates female fertility and promotes CsRALF4/19 expression in the ovary during pollen tube guidance in cucumber.

Overexpressing Vitamin C Defective 2 reduces fertility and alters Ca2+ signals in Arabidopsis pollen.

A potential strategy to mitigate oxidative damage in plants is to increase the abundance of antioxidants, such as ascorbate (i.e. vitamin C). In Arabidopsis (A. thaliana), a rate-limiting step in ascorbate biosynthesis is a phosphorylase encoded by Vitamin C Defective 2 (VTC2). To specifically overexpress VTC2 (VTC2 OE) in pollen, the coding region was expressed using a promoter from a gene with ∼150-fold higher expression in pollen, leading to pollen grains with an eight-fold increased VTC2 mRNA. VTC2 OE resulted in a near-sterile phenotype with a 50-fold decrease in pollen transmission efficiency and a five-fold reduction in the number of seeds per silique. In vitro assays revealed pollen grains were more prone to bursting (greater than two-fold) or produced shorter, morphologically abnormal pollen tubes. The inclusion of a genetically encoded Ca2+ reporter, mCherry-GCaMP6fast (CGf), revealed pollen tubes with altered tip-focused Ca2+ dynamics and increased bursting frequency during periods of oscillatory and arrested growth. Despite these phenotypes, VTC2 OE pollen failed to show expected increases in ascorbate or reductions in reactive oxygen species, as measured using a redox-sensitive dye or a roGFP2. However, mRNA expression analyses revealed greater than two-fold reductions in mRNA encoding two enzymes critical to biosynthetic pathways related to cell walls or glyco-modifications of lipids and proteins: GDP-d-mannose pyrophosphorylase (GMP) and GDP-d-mannose 3',5' epimerase (GME). These results support a model in which the near-sterile defects resulting from VTC2 OE in pollen are associated with feedback mechanisms that can alter one or more signaling or metabolic pathways critical to pollen tube growth and fertility.