Central-cell-produced attractants control fertilization recovery.

Animal fertilization relies on hundreds of sperm racing toward the egg, whereas, in angiosperms, only two sperm cells are delivered by a pollen tube to the female gametes (egg cell and central cell) for double fertilization. However, unsuccessful fertilization under this one-pollen-tube design can be detrimental to seed production and plant survival. To mitigate this risk, unfertilized-gamete-controlled extra pollen tube entry has been evolved to bring more sperm cells and salvage fertilization. Despite its importance, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we report that, in Arabidopsis, the central cell secretes peptides SALVAGER1 and SALVAGER2 in a directional manner to attract pollen tubes when the synergid-dependent attraction fails or is terminated by pollen tubes carrying infertile sperm cells. Moreover, loss of SALs impairs the fertilization recovery capacity of the ovules. Therefore, this research uncovers a female gamete-attraction system that salvages seed production for reproductive assurance.

Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance.

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

SPA, a Stigma-style-transmitting tract Physical microenvironment Assay for investigating mechano-signaling in pollen tubes.

Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it's naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.

Arabidopsis VILLIN5 bundles actin filaments using a novel mechanism.

Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments.

In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

Myo-inositol oxygenase CgMIOX3 alleviates S-RNase-induced inhibition of incompatible pollen tubes in pummelo.

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 down-regulation 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 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.

Embryo sac development relies on symplastic signals from ovular integuments in Arabidopsis.

Ovules are female reproductive organs of angiosperms, consisting of sporophytic integuments surrounding female gametophytes, that is, embryo sacs. Synchronization between integument growth and embryo sac development requires intracellular communication. However, signaling routes through which cells of the two generations communicate are unclear. We report that symplastic signals through plasmodesmata (PDs) of integuments are critical for the development of female gametophytes. Genetic interferences of PD biogenesis either by functional loss of CHOLINE TRANSPORTER-LIKE1 (CTL1) or by integument-specific expression of a mutated CALLOSE SYNTHASE 3 (cals3m) compromised PD formation in integuments and reduced fertility. Close examination of pINO:cals3m or ctl1 ovules indicated that female gametophytic development was either arrested at various stages after the formation of functional megaspores. In both cases, defective ovules could not attract pollen tubes, leading to the failure of fertilization. Results presented here demonstrate a key role of the symplastic route in sporophytic control of female gametophytic development.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Arabidopsis Sar1b is critical for pollen tube growth.

Pollen tube growth is an essential step leading to reproductive success in flowering plants, in which vesicular trafficking plays a key role. Vesicular trafficking from endoplasmic reticulum to the Golgi apparatus is mediated by the coat protein complex II (COPII). A key component of COPII is small GTPase Sar1. Five Sar1 isoforms are encoded in the Arabidopsis genome and they show distinct while redundant roles in various cellular and developmental processes, especially in reproduction. Arabidopsis Sar1b is essential for sporophytic control of pollen development while Sar1b and Sar1c are critical for gametophytic control of pollen development. Because functional loss of Sar1b and Sar1c resulted in pollen abortion, whether they influence pollen tube growth was unclear. Here we demonstrate that Sar1b mediates pollen tube growth, in addition to its role in pollen development. Although functional loss of Sar1b does not affect pollen germination, it causes a significant reduction in male transmission and of pollen tube penetration of style. We further show that membrane dynamics at the apex of pollen tubes are compromised by Sar1b loss-of-function. Results presented provide further support of functional complexity of the Sar1 isoforms.

The PPO family in Nicotiana tabacum is an important regulator to participate in pollination.

Polyphenol oxidases (PPOs) are type-3 copper enzymes and are involved in many biological processes. However, the potential functions of PPOs in pollination are not fully understood. In this work, we have screened 13 PPO members in Nicotiana. tabacum (named NtPPO1-13, NtPPOs) to explore their characteristics and functions in pollination. The results show that NtPPOs are closely related to PPOs in Solanaceae and share conserved domains except NtPPO4. Generally, NtPPOs are diversely expressed in different tissues and are distributed in pistil and male gametes. Specifically, NtPPO9 and NtPPO10 are highly expressed in the pistil and mature anther. In addition, the expression levels and enzyme activities of NtPPOs are increased after N. tabacum self-pollination. Knockdown of NtPPOs would affect pollen growth after pollination, and the purines and flavonoid compounds are accumulated in self-pollinated pistil. Altogether, our findings demonstrate that NtPPOs potentially play a role in the pollen tube growth after pollination through purines and flavonoid compounds, and will provide new insights into the role of PPOs in plant reproduction.

Integrative phenotyping analyses reveal the relevance of the phyB-PIF4 pathway in Arabidopsis thaliana reproductive organs at high ambient temperature.

BACKGROUND: The increasing ambient temperature significantly impacts plant growth, development, and reproduction. Uncovering the temperature-regulating mechanisms in plants is of high importance, for increasing our fundamental understanding of plant thermomorphogenesis, for its potential in applied science, and for aiding plant breeders in improving plant thermoresilience. Thermomorphogenesis, the developmental response to warm temperatures, has been primarily studied in seedlings and in the regulation of flowering time. PHYTOCHROME B and PHYTOCHROME-INTERACTING FACTORs (PIFs), particularly PIF4, are key components of this response. However, the thermoresponse of other adult vegetative tissues and reproductive structures has not been systematically evaluated, especially concerning the involvement of phyB and PIFs. RESULTS: We screened the temperature responses of the wild type and several phyB-PIF4 pathway Arabidopsis mutant lines in combined and integrative phenotyping platforms for root growth in soil, shoot, inflorescence, and seed. Our findings demonstrate that phyB-PIF4 is generally involved in the relay of temperature signals throughout plant development, including the reproductive stage. Furthermore, we identified correlative responses to high ambient temperature between shoot and root tissues. This integrative and automated phenotyping was complemented by monitoring the changes in transcript levels in reproductive organs. Transcriptomic profiling of the pistils from plants grown under high ambient temperature identified key elements that may provide insight into the molecular mechanisms behind temperature-induced reduced fertilization rate. These include a downregulation of auxin metabolism, upregulation of genes involved auxin signalling, miRNA156 and miRNA160 pathways, and pollen tube attractants. CONCLUSIONS: Our findings demonstrate that phyB-PIF4 involvement in the interpretation of temperature signals is pervasive throughout plant development, including processes directly linked to reproduction.

Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

The transcription factor LBD10 sustains pollen tube growth and integrity by modulating reactive oxygen species homeostasis via the regulation of flavonol biosynthesis in Arabidopsis.

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.

Reactive oxygen species are signaling molecules that modulate plant reproduction.

Reactive oxygen species (ROS) can serve as signaling molecules that are essential for plant growth and development but abiotic stress can lead to ROS increases to supraoptimal levels resulting in cellular damage. To ensure efficient ROS signaling, cells have machinery to locally synthesize ROS to initiate cellular responses and to scavenge ROS to prevent it from reaching damaging levels. This review summarizes experimental evidence revealing the role of ROS during multiple stages of plant reproduction. Localized ROS synthesis controls the formation of pollen grains, pollen-stigma interactions, pollen tube growth, ovule development, and fertilization. Plants utilize ROS-producing enzymes such as respiratory burst oxidase homologs and organelle metabolic pathways to generate ROS, while the presence of scavenging mechanisms, including synthesis of antioxidant proteins and small molecules, serves to prevent its escalation to harmful levels. In this review, we summarized the function of ROS and its synthesis and scavenging mechanisms in all reproductive stages from gametophyte development until completion of fertilization. Additionally, we further address the impact of elevated temperatures induced ROS on impairing these reproductive processes and of flavonol antioxidants in maintaining ROS homeostasis to minimize temperature stress to combat the impact of global climate change on agriculture.

The emerging role of cysteine-rich peptides in pollen-pistil interaction.

Unlike early land plants, flowering plants have evolved a pollen tube that transport a pair of non-motile sperm cells to the female gametophyte. This process, known as siphonogamy, was first observed in gymnosperms and later become prevalent in angiosperms. However, the precise molecular mechanisms underlying the male-female interactions remain enigmatic. From the pollen grain's landing on the stigma to gametes fusion, the male part needs to pass various tests: How does the stigma distinguish between compatible and incompatible pollen? What mechanisms guide pollen tube towards the ovule? What factors trigger pollen tube rupture? How is polyspermy prevented? And how does the sperm cell ultimately reach the egg? Successful male-female communications is essential for surmounting these challenges, with cysteine-rich peptides (CRPs) playing a pivotal role in these dialogues. In this review, we summarize the characteristics of four distinct classes of CRPs and then we systematically review the recent progresses of the role of CRPs in four crucial stages of pollination and fertilization. Finally, we conclude by considering the potential applications of this knowledge in crop breeding, and suggesting avenues for future research.

Heat stress and sexual reproduction in maize: unveiling the most pivotal factors and the greatest opportunities.

The escalation in the intensity, frequency, and duration of high-temperature (HT) stress is currently unparalleled, which aggravates the challenges for crop production. Yet, the stage-dependent responses of reproductive organs to HT stress at the morphological, physiological, and molecular levels remain inadequately explored in pivotal staple crops. This review synthesized current knowledge regarding the mechanisms by which HT stress induces abnormalities and aberrations in reproductive growth and development, as well as by which it alters the morphology and function of florets, flowering patterns, and the processes of pollination and fertilization in maize (Zea mays L.). We identified the stage-specific sensitivities to HT stress and accurately defined the sensitive period from a time scale of days to hours. The microspore tetrad phase of pollen development and anthesis (especially shortly after pollination) are most sensitive to HT stress, and even brief temperature spikes during these stages can lead to significant kernel loss. The impetuses behind the heat-induced impairments in seed set are closely related to carbon, reactive oxygen species, phytohormone signals, ion (e.g. Ca2+) homeostasis, plasma membrane structure and function, and others. Recent advances in understanding the genetic mechanisms underlying HT stress responses during maize sexual reproduction have been systematically summarized.

The fertility tracks of pollen tube in the ovary of Solanum nigrum by three-dimensional reconstruction.

In this paper, we present an enhanced method for automatically capturing a large number of consecutive paraffin sections using a microscope. Leveraging these microstructural images, we employed three-dimensional visualisation and reconstruction techniques to investigate the dispersal growth process of pollen tube bundles upon entering the ovary of Solanum nigrum. Additionally, we explored their behaviour within different ovules and examined the relationship between the germination rate of seeds and the fertilisation process. Our findings reveal that despite the abundance of Solanum nigrum seeds, only a fraction of them is capable of successful germination. The germination rate of seeds is closely related to whether fertilisation of the ovules and pollen tubes is completed. Due to the limited number of pollen tubes entering the ovary, only a portion of the ovules can be fertilised. The proportion of fertilised ovules positively correlates with the germination rate of the seeds. Through three-dimensional reconstruction, we observed a phenomenon of proximity during the pollination process, wherein ovules closer to the pollen tube bundles are more likely to be fertilised. Furthermore, fertilised ovules exhibited significant changes in morphology and embryo sac structure. The number of fertilised ovules directly impacts the germination rate of wild Solanum nigrum seeds. Although all Solanum nigrum ovules have the potential to develop into seeds, most seeds originating from unfertilised ovules are unable to germinate normally, resulting in an incomplete germination rate of seeds and preventing it from reaching 100%.

Screening methods for thermotolerance in pollen.

Plant reproduction is highly susceptible to temperature stress. The development of the male gametophyte in particular represents a critical element in the reproductive cycle with high sensitivity to elevated temperatures. Various methods have been used to test the effect of temperature stress on pollen performance or to determine the degree of susceptibility of given species and genotypes. The information gained informs the development of new crop varieties suited to grow under warmer conditions arising through climate change and facilitates predicting the behavior of natural populations under these conditions. The characterization of pollen performance typically employs the terms pollen viability and pollen vigor, which, however, are not necessarily used consistently across studies. Pollen viability is a nominal parameter and is often assayed relying on cellular features as proxy to infer the capability of pollen grains to germinate and complete double fertilization. Alternatively, pollen germination can be determined through in vitro growth assays, or by monitoring the ability of pollen tubes to complete different progamic steps in vivo (ability to reach an ovule, release sperm cells, lead to seed set). Pollen vigor is an ordinal parameter that describes pollen tube growth rate or the efficiency of pollen tube growth as inferred by its morphology or growth pattern. To ensure consistent and relevant terminology, this review defines these terms and summarizes the methodologies used to assess them.

Floral freezing tolerance is tied to flowering time in North American woody plant species.

BACKGROUND AND AIMS: As winter and spring temperatures continue to increase, the timing of flowering and leaf out is advancing in many seasonally cold regions. This advancement could put plants that flower early in the spring at risk of decreased reproduction in years when there are late freeze events. Unfortunately, relatively little is known about floral freezing tolerance in forest communities. In this study, we examined the impact of freezing temperatures on the flowers of woody plants in a region where there is rapid winter warming in North America. METHODS: We subjected the flowers of twenty-five woody species to a hard (-5ºC) and a light freeze (0ºC). We assessed tissue damage using electrolyte leakage. In a subset of species, we also examined the impact of a hard freeze on pollen tube growth. To determine if the vulnerability of flowers to freezing damage relates to flowering time and to examine the responsiveness of flowering time to spring temperature, we recorded the date of first flower for our study species for three years. KEY RESULTS AND CONCLUSIONS: Across species, we found that floral freezing tolerance was strongly tied to flowering time with the highest freezing tolerance occurring in plants that bloomed earlier in the year. We hypothesize that these early blooming species are unlikely to be impacted by a false spring. Instead, the most vulnerable species to a false spring should be those that bloom later in the season. The flowering time in these species is also more sensitive to temperature, putting them at a great risk of experiencing a false spring. Ultimately, floral damage in one year will not have a large impact on species fitness, but if false springs become more frequent, there could be long-term impacts on reproduction of vulnerable species.

Temperature dependence of pollen germination and tube growth in conifers relates to their distribution along an elevational gradient in Washington State, USA.

BACKGROUND AND AIMS: Pollen germination and tube growth are essential processes for successful fertilization. They are among the most temperature-vulnerable stages and subsequently affect seed production and determine population persistence and species distribution under climate change. Our study aims to investigate intra- and inter-specific variations in the temperature dependence of pollen germination and tube length growth and to explore how these variations differ for pollen from elevational gradients. METHODS: We focused on three conifer species, Pinus contorta, Picea engelmannii, and Pinus ponderosa, with pollen collected from 350 to 2200m elevation in Washington State, USA. We conducted pollen viability tests at temperatures from 5 to 40°C in 5°C intervals. After testing for four days, we took images of these samples under a microscope to monitor pollen germination percentage (GP) and tube length (TL). We applied the Gamma function to describe the temperature dependence of GP and TL and estimated key parameters, including the optimal temperature for GP (Topt_GP) and TL (Topt_TL). KEY RESULTS: Results showed that pollen from three species and different elevations within a species have different GP, TL, Topt_GP, and Topt_TL. The population with a higher Topt_GP would also have a higher Topt_TL, while Topt_TL was generally higher than Topt_GP, i.e., a positive but not one-to-one relationship. However, only Pinus contorta showed that populations from higher elevations have lower Topt_GP and Topt_TL and vice versa. The variability in GP increased at extreme temperatures, whereas the variability in TL was greatest near Topt_TL. CONCLUSIONS: Our study demonstrates the temperature dependences of three conifers across a wide range of temperatures. Pollen germination and tube growth are highly sensitive to temperature conditions and vary among species and elevations, affecting their reproduction success during warming. Our findings can provide valuable insights to advance our understanding of how conifer pollen responds to rising temperatures.