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Background Polyploidization plays a major role in citrus breeding, particularly in generating triploid hybrids through controlled crosses between diploid female and tetraploid male parents. However, limited information is available on how polyploidy affects pollen performance in citrus, and in particular, how temperature influences these processes. In this study, we compared pollen viability under field conditions and pollen tube growth at different temperatures in diploid and tetraploid genotypes, and assessed whether the observed differences were related to genomic instability caused by the tetraploidization treatment. Results Tetraploid `Clemenules´ and `Fortune´, both colchicine-derived, showed a higher proportion of collapsed pollen grains and reduced viability compared with their diploid counterparts, whereas the spontaneous tetraploid `Pineapple´ displayed values similar to those of the diploid. Pollen tube growth assays revealed slightly faster tube elongation in tetraploids, and notably, self-incompatible diploid pistils of `Fortune´ were successfully fertilized by pollen from its tetraploid form. Genotyping-by-sequencing (GBS) analyses of diploid and tetraploid plants revealed no chromosomal rearrangements or dosage alterations, suggesting that colchicine treatment did not induce large-scale genomic instability. Conclusions Our findings demonstrate that tetraploidy influences several aspects of citrus pollen performance, while genomic stability remains largely preserved in colchicine-induced genotypes. These results provide new insights into the reproductive biology of citrus polyploids under different temperature scenarios and reinforce the potential use of tetraploid parents in triploid breeding programs. Key message Pollen grains of diploid and tetraploid citrus genotypes show a different performance, while there is no evidence of chromosomal instability during the tetraploidization process.

Main conclusionSelf-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.AbstractssApples [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.

In flowering plants, the success of fertilization depends on the rapid polar extension of a pollen tube, which delivers sperm cells to the female gametophyte for fertilization. Numerous studies have shown that the microenvironment in planta is more conducive to the growth and development of pollen tubes than that in vitro. However, how stigma factors coordinate to regulate pollen tube growth is still poorly understood. Here, we demonstrate that in tobacco, mature stigma extract, but not immature stigma extract, facilitates pollen tube growth. Comparative transcriptomic and qRT-PCR analyses showed that the differentially expressed genes during stigma maturation were mainly enriched in the metabolism pathway. Through metabolome analyses, about 500 metabolites were identified to be differently accumulated; the significantly increased metabolites in the mature stigmas mainly belonged to alkaloids, flavonoids, and terpenoids, while the downregulated differential metabolites were related to lipids, amino acids, and their derivatives. Among the different kinds of plant hormones, the cis-form contents of zeatin were significantly increased, and more importantly, cis-zeatin riboside promoted pollen tube growth in vitro. Thus, our results reveal an overall landscape of gene expression and a detailed nutritional microenvironment established for pollen tube growth during the process of stigma maturation, which provides valuable clues for optimizing in vitro pollen growth and investigating the pollen–stigma interaction.

Agriculture, particularly fruit production, is considered a crucial industry with a significant economic impact in many countries. Extreme fluctuations in air temperature can negatively affect the flowering periods of fruit species. Therefore, it is important to conduct studies on pollen performance analysis to determine these effects. Pollen performance analysis has seen significant advancements in agricultural research, with the emergence of new pollen germination methods driven by advances in technology and equipment. In these analyses, in addition to traditional approaches, Artificial Neural Networks and deep learning have gained importance recently. The main objective of this study is to develop a model that predicts the germination rate based on a given set of input variables. Firstly, pollen germination rate and pollen tube length were determined in vitro. Pollen grains from four cultivars of pome fruit were sown in three different media and incubated for four different durations at seven different temperatures for an in vitro test. Three deep-learning models with two hidden layers were developed and different optimizers were considered for model development. The best model was selected through a validation test.This study aimed to develop a machine learning model for predicting pollen germination rates in pome fruits. Pollen grains from four cultivars were subjected to in vitro germination tests under varying temperatures, media, and durations. Using artificial neural networks, the model achieved an R² value of 0.89 with the Adam optimizer, demonstrating high accuracy in predicting germination rates. These findings highlight the potential of machine learning in advancing agricultural research.

Aspartic proteases (APs) comprise a large proteolytic enzyme family widely distributed in animals, microbes, viruses, and plants. The rice genome encodes 96 APs, of which only a few have been functionally characterized. Here, the identification and characterization of a novel AP gene, OsAP65, which plays an indispensable role in pollen tube growth in rice, is reported. The T-DNA insertion line of OsAP65 caused severe segregation distortion. In the progeny derived from an individual heterozygous for the T-DNA insertion, the wild type and T-DNA-carrying heterozygote segregated at a ratio close to 1:1, while homozygotes of disrupted OsAP65 (OsAP65–/–) were not recovered. Reciprocal crosses between heterozygotes and wild-type plants demonstrated that the mutant alleles could not be transmitted through the male gamete. Examination of the anthers from heterozygous plants revealed that the mutant pollen matured normally, but did not germinate or elongate. OsAP65 was expressed in various tissues and the transcript level in heterozygous plants was about half of the amount measured in the wild-type plants. The subcellular localization showed that OsAP65 is a pre-vacuolar compartment (PVC) protein. These results indicated that OsAP65 was essential for rice pollen germination and tube growth.

The establishment of pollen–pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen–stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen–stigma interaction. Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP-B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth. Our results revealed that pollen hydration is severely impaired when multiple PCP-Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied. These results demonstrate that AtPCP-Bs are key regulators of the hydration ‘checkpoint’ in establishment of pollen–stigma compatibility. In addition, we propose that interspecies diversity of PCP-Bs may contribute to reproductive barriers in the Brassicaceae.

Leu-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal Leu-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis (Arabidopsis thaliana). Mutations in multiple pollen-expressed lrx genes cause severe defects in pollen germination and pollen tube growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca²⁺ availability partially suppresses the pollen tube growth defects, suggesting that LRX proteins influence Ca²⁺-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modeling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

Global warming intensifies extreme heat events, threatening crop reproduction by impairing pollen development, germination, and tube growth. However, the mechanisms underlying pollen heat responses remain elusive. The actin cytoskeleton and actin-binding proteins (ABPs) are crucial in these processes, yet their roles under heat stress are poorly understood. Here, we identify a mutant, pollen germination sensitive to LatB (pgsl1 ), via forward genetic screening. PGSL1 encodes a heat-stable, plant-specific ABP that binds and stabilizes actin filaments (F-actin), preventing heat-induced denaturation. High temperatures reduce F-actin content but promote bundling in pollen tubes. Notably, pgsl1 mutants exhibit decreased F-actin abundance and bundling under heat stress compared to wild-type plants. These findings highlight PGSL1 as a key regulator of actin dynamics, essential for pollen heat tolerance, offering potential strategies to enhance crop resilience in a warming climate. The actin cytoskeleton plays crucial roles in pollen development, germination, and tube growth. Here the authors show that PGSL1, an Arabidopsis heat-stable actin binding protein stabilizes actin filaments under high temperatures, preventing denaturation and maintaining F-actin dynamics.

The vegetative cell nucleus proceeds ahead of a pair of sperm cells located beneath the pollen tube tip during germination. The tip-localized vegetative nucleus had been considered to play a pivotal role in the control of directional pollen tube growth and double fertilization. However, we recently reported the female-targeting behavior of pollen tubes from mutant plants, of which the vegetative nucleus and sperm nuclei were artificially immotile. We showed that the apical region of the mutant pollen tubes became physiologically enucleated after the first callose plug formation, indicating the autonomously growing nature of pollen tubes without the vegetative nucleus and sperm cells. Thus, in this study, we further analyzed another Arabidopsis thaliana mutant producing physiologically enucleated pollen tubes and discussed the mechanism by which a pollen tube can grow without de novo transcription from the vegetative nucleus. We propose several possible molecular mechanisms for persistent pollen tube growth, such as the contribution of transcripts before and immediately after germination and the use of persistent transcripts, which may be important for a competitive race among pollen tubes.

During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity.