Pollen Tube Growth Research Articles

Identification and characterization of photo-thermo insensitive cowpea (Vigna unguiculata) genotypes for hot arid environment

High green pod yield, dual purpose type, earliness and photo-thermo insensitivity are the major breeding objectives of cowpea [Vigna unguiculata (L.) Walp.]. Photo-thermo sensitivity makes this crop vulnerable to photoperiod and temperature fluctuations particularly in hot arid regions, thereby affecting its yield potential drastically. Therefore, the present study was aimed to identify such elite genotypes with photo-thermo insensitivity, which can fit well across all seasons under hot arid environment. The experiments were conducted at ICAR-Central Institute for Arid Horticulture, Bikaner during rainy and summer seasons of 2019–20 and 2020–21. A wide range of variability among genotypes was observed with respect to various phenological and yield traits over the environments. Among the evaluated genotypes, AHCP-1-4-1 and AHCP-2-3 were found photo-thermo insensitive as these were able to flower and set pods at temperatures as low as 10oC and as high as 46°C. Pollen viability studies indicated that AHCP-1-4-1 had 83.72 and 88.24% pollen viability and AHCP-2-3 had 81.58 and 85.71% viable pollen at 11°C and 46°C, respectively and normal pollen tube growth at both the extremes of temperature. The identified genetic resources will contribute for developing photo-thermo insensitive cultivars and will improve the productivity and extend the availability of cowpea with quality pod yield across all seasons and locations of hot arid regions.

Floral Development on Vitis vinifera Is Associated with MADS-Box Transcription Factors through the Transcriptional Regulation of VviZIP3.

Several grapevine (Vitis vinifera L.) cultivars show a tendency to develop parthenocarpic seedless grapes, affecting fruit yield and quality. This reproductive disorder originates in defective ovule fertilization due to a failure in pollen tube growth. Zinc (Zn) is a crucial trace element, playing a vital role in various physiological and metabolic processes. It is particularly essential for the healthy growth of flowers and fruits. Insufficient zinc has been suggested as a potential reason for issues in this development process. This microelement is taken up through a mechanism that involves transporters, including the ZRT-IRT-like protein (ZIP) gene family, associated with the influx of Zn into the cell. In grapevines, 20 genes for ZIP-type transporters have been described. In this study, we analyzed the expression pattern of VviZIP3 during flower development and employ transgenic methods to assess its transcriptional regulation. Furthermore, through computational examination of the promoter region, we identified two CArG boxes, recognized as responsive elements to MADS transcription factors. These factors play a key role in shaping various components of a flower, such as pollen. Our investigation of the VviZIP3 promoter confirms the functionality of these CArG boxes. Overall, our results suggest that the increased expression of VviZIP3 during flowering is likely under the influence of MADS transcription factors.

Breeding system in Olea ferruginea Royle (Oleaceae): comparative performance of staminate and perfect flowers in reproductive assurance

ABSTRACT Olea ferruginea, a wild widely distributed species of the olive lineage, exhibits andromonoecy with protogynous perfect (hermaphrodite) and staminate (male) flowers. The present communication elucidates the differential performance of staminate and perfect flowers in reproductive assurance. The flowering chronology and function of perfect and staminate flowers in the breeding system were investigated to understand the breeding behavior of the species. In terms of breeding behavior, pollen from staminate and perfect flowers was used to establish their role in breeding system under different pollination treatments. Results indicate higher pollen viability, germination, and pollen tube growth of staminate than perfect flowers. Results from pollen counts and morphological investigations show slight differences in both types. The species is self-incompatible which is indicated by pollen germination, the average number of pollen tubes in the style, and the fruit set following self and cross-pollination. Hand cross-pollination improves natural fruit set, especially when pollen from staminate flowers was used. Although the relict role of attracting pollinators cannot be fully discarded, it is suggested that the main benefit supplied by the formation of staminate flowers in olives is to promote male fitness by raising pollen output at the population level.

Compounds composition of pollen tubes of Scots pine (Pinus sylvestris L.).

The aim of this research was to study the composition of pollen tubes of Scots pine (Pinus sylvestris L.). Pollen cultivation on deionized distilled water excluded the potential influence of the cultivation medium on the pollen tube growth and development. The fluorescent study indicated a gradual distribution of chemical compounds along the length of the tube. It was shown that the protoplast apical zone and the parietal layer near the tube's tip are most likely actively involved in the ion transport regulation in the growing pollen tube. The callose synthesis in the tip of matured pine tube completed the first stage of its active growth. Significant differences and pH gradients at the nucleus region and the parietal layer of the tube wall indicate that H+ gradient is the direct driving force of vesicle transport and can regulate the growth of pollen tubes. The distribution of amino acids, RNA, proteins and lipids was uniform throughout the length of the pine pollen tube. The content of amino acids, RNA, DNA and proteins slightly increased near the cell nucleus and drastically increased in the apical zone. At the very tip of the tube, a slight increase in the concentration of polysaccharides and a significant decrease in the content of amino acids, RNA, DNA, proteins and lipids were detected.

Reproductive systems and hybridization of Lymania species (Bromeliaceae) endemic to Northeast Brazil threatened with extinction

Reproductive systems among the Bromeliaceae family are very diverse at the species level, including allogamous, autogamous, cleistogamous, agamospermic or mixed plants. Hybridization allows the creation of new genotypes with different combinations of shapes and colors to meet the ornamental plant market's demand for novelty. Knowing the reproductive strategies of the species makes it possible to obtain these new materials with ornamental potential, in addition to being essential to define better conservation strategies. Little is known about the reproductive biology of Lymania species. In addition, at least 60% of these species are on the Brazilian National List of Species Threatened with Extinction. The purpose of this study was to identify the reproductive systems, the possible interspecific hybridizations, and the possible reproductive barriers of nine Lymania species based on the germination of pollen grains and the corresponding growth of the pollen tube in the pistil by using fluorescence microscopy with ultraviolet filtering. Natural and controlled pollinations of the nine species were carried out. The reproductive strategy was defined according to the autogamy index (AI), self-incompatibility index (SII), and reproductive efficiency index (REI). All species were self-incompatible except for L. smithii, which was autogamous. Agamospermy was not observed in any of the studied species. Forty-one hybridization combinations between species were performed, resulting in a 54% success rate, with incongruity in only 12 combinations. The main incongruence phenomena identified after interspecific crosses were: poor germination of pollen grains on the stigma surface; inhibition of pollen tube growth along the style; coiling of pollen tubes along the style; pollen tubes inside the ovary but without penetration into the micropyle; and reticulate deposition of callose in the pollen tube, among others. We suggest that self-incompatibility of the species is gametophytic, preventing self-fertilization. Results showed that it is possible to perform hybridization with some species. However, morphological, genetic, and molecular studies are needed to confirm the potential of these species. Seeds generated in the different reproductive systems, as well as in the hybridizations, had germination rates above 94%. The results presented here make important contributions to the conservation of species, seed generation, and hybridization of Bromeliaceae.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

SD-RLK28 positively regulates pollen hydration on stigmas as a PCP-Bβ receptor in Arabidopsis thaliana.

Pollen hydration on dry stigmas is strictly regulated by pollen-stigma interactions in Brassicaceae. Although several related molecular events have been described, the molecular mechanism underlying pollen hydration remains elusive. Multiple B-class pollen coat proteins (PCP-Bs) are involved in pollen hydration. Here, by analyzing the interactions of two PCP-Bs with three Arabidopsis thaliana stigmas strongly expressing S-domain receptor kinase (SD-RLK), we determined that SD-RLK28 directly interacts with PCP-Bβ. We investigated pollen hydration, pollen germination, pollen tube growth, and stigma receptivity in the sd-rlk28 and pcp-bβ mutants. PCP-Bβ acts in the pollen to regulate pollen hydration on stigmas. Loss of SD-RLK28 had no effect on pollen viability, and sd-rlk28 plants had normal life cycles without obvious defects. However, pollen hydration on sd-rlk28 stigmas was impaired and pollen tube growth in sd-rlk28 pistils was delayed. The defect in pollen hydration on sd-rlk28 stigmas was independent of changes in reactive oxygen species levels in stigmas. These results indicate that SD-RLK28 functions in the stigma as a PCP-Bβ receptor to positively regulate pollen hydration on dry stigmas.

Heat-dependent postpollination limitations on maize pollen tube growth and kernel sterility.

Heat stress has a negative impact on pollen development in maize (Zea mays L.) but the postpollination events that determine kernel sterility are less well characterised. The impact of short-term (hours) heat exposure during postpollination was therefore assessed in silks and ovaries. The temperatures inside the kernels housed within the husks was significantly lower than the imposed heat stress. This protected the ovaries and possibly the later phase of pollen tube growth from the adverse effects of heat stress. Failure of maize kernel fertilization was observed within 6 h of heat stress exposure postpollination. This was accompanied by a significant restriction of early pollen tube growth rather than pollen germination. Limitations on early pollen tube growth were therefore a major factor contributing to heat stress-induced kernel sterility. Exposure to heat stress altered the sugar composition of silks, suggesting that hexose supply contributed to the limitations on pollen tube growth. Moreover, the activities of sucrose metabolising enzymes, the expression of sucrose degradation and trehalose biosynthesis genes were decreased following heat stress. Significant increases in reactive oxygen species, abscisic acid and auxin levels accompanied by altered expression of phytohormone-related genes may also be important in the heat-induced suppression of pollen tube growth.

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.

Transcriptomic Insight into the Pollen Tube Growth of Olea europaea L. subsp. europaea Reveals Reprogramming and Pollen-Specific Genes Including New Transcription Factors.

The pollen tube is a key innovation of land plants that is essential for successful fertilisation. Its development and growth have been profusely studied in model organisms, but in spite of the economic impact of olive trees, little is known regarding the genome-wide events underlying pollen hydration and growth in this species. To fill this gap, triplicate mRNA samples at 0, 1, 3, and 6 h of in vitro germination of olive cultivar Picual pollen were analysed by RNA-seq. A bioinformatics R workflow called RSeqFlow was developed contemplating the best practices described in the literature, covering from expression data filtering to differential expression and clustering, to finally propose hub genes. The resulting olive pollen transcriptome consisted of 22,418 reliable transcripts, where 5364 were differentially expressed, out of which 173 have no orthologue in plants and up to 3 of them might be pollen-specific transcription factors. Functional enrichment revealed a deep transcriptional reprogramming in mature olive pollen that is also dependent on protein stability and turnover to allow pollen tube emergence, with many hub genes related to heat shock proteins and F-box-containing proteins. Reprogramming extends to the first 3 h of growth, including processes consistent with studies performed in other plant species, such as global down-regulation of biosynthetic processes, vesicle/organelle trafficking and cytoskeleton remodelling. In the last stages, growth should be maintained from persistent transcripts. Mature pollen is equipped with transcripts to successfully cope with adverse environments, even though the in vitro growth seems to induce several stress responses. Finally, pollen-specific transcription factors were proposed as probable drivers of pollen germination in olive trees, which also shows an overall increased number of pollen-specific gene isoforms relative to other plants.

Role of Phenylpropanoids and Flavonoids in Plant Defense Mechanism

Phenylpropanoids and flavonoids are specialized metabolites that play a crucial role in plant defenses against biotic and abiotic stresses. Secondary metabolites in plants, such as phenylpropanoids, flavonoids, lignins, monolignols, phenolic acids, stilbenes and coumarins, play a significant role in biotic and abiotic stress responses and interactions with their environment. Under stress-free conditions, flavonoids influence pollen tube growth, seed maturation, dormancy, and the longevity of plant reproductive organs and seeds. Under adverse abiotic conditions, flavonoids can mediate defense responses, such as the accumulation of flavonoids in leaves and glandular trichomes. Metabolomics has identified specialized PPP metabolites correlated with plant resistance to fungi and oomycetes. However, the correlation can be in opposite directions depending on the pathogen and a systemic approach is required to achieve increased resistance without decreasing resistance to another pathogen. The PAL gene is located upstream in the PPP pathway and phenylpropanoids and flavonoids have antifungal activity against Fusarium oxysporum, Rhizoctonia solani and Alternaria alternata. Phenylpropanoids have been studied in vitro to develop biological alternatives to synthetic phytoprotectants but the host's defense system is the most effective mode of action. Post-inoculation transcriptomic shifts reveal that some genes encoding enzymes in the PPP are rapidly activated, while others are induced later in the infection response. Insects are the most problematic macroscopic pests on cultivated plants and plants' natural defense mechanisms against herbivores include releasing volatile organic compounds, attracting enemies and producing phenylpropanoids and flavonoids. Increasing the content of flavonoids in infested plants can reduce the population of insects that cause damage to plants and spread viruses. Plant-environment interaction, mediated by secondary metabolites like phenylpropanoids and flavonoids, leads to diversity in gene structures. Systemic biology approach will be realized through sequencing, structural elucidation, and analytical tools. Model plants and crops can be used for exploration.

Cytogenetic Study and Pollen Viability of Phalaenopsis Queen Beer 'Mantefon'.

Intergeneric and interspecific hybridization has been employed for the breeding of Phalaenopsis to transfer desirable traits between species, producing novel phenotypes with improved size, color, form, and flower-bearing ability. These characteristics are often enhanced; however, many of these hybrids are triploids and have reduced or complete sterility, for example, Phalaenopsis Queen Beer 'Mantefon', an important novelty-type cultivar in Asia, particularly in China, Japan, and Republic of Korea. Despite the increasing demand for the crop for ornamental purposes, little is known about its cytogenetics, which is essential for breeding and, consequently, crop improvement. In this study, karyotyping using fluorescence in situ hybridization, meiotic chromosome behavior analysis, pollen staining, and in vitro viability germination tests were performed to understand the cause of hybrid sterility and pollen abnormality in Phalaenopsis Queen Beer 'Mantefon' from a cytogenetic perspective. Viability tests revealed pollen infertility at all flower developmental stages, confirmed by the absence of pollen tube growth. Aberrant chromosomal behavior was observed in pollen mother cells (PMCs), frequently forming univalents, chromosomal bridges, and laggards during the entire meiotic process. PMCs were also divided irregularly into sporads with varying numbers of micronuclei, which may be responsible for pollen sterility in this cultivar. Altogether, the cytogenetic analyses provided insights into the pollen development of Phalaenopsis Queen Beer 'Mantefon' and the conceivable causes of its infertility.

Identification and analysis of CdS-RNase in Camellia drupifera: A key determinant of late-acting self-incompatibility

Plant self-incompatibility (SI) is of great significance in reproductive biology and is also the main factor affecting yield in agricultural production. Late-acting self-incompatibility (LSI) is one of the main types of plant SI, but the mechanism of LSI is not systematically analyzed. Tea oil Camellia, a strictly outcrossing plant, is a well-known woody oil cash crop. SI is one of the main reasons for the low yield of tea oil Camellia. In this study, we found that C. drupifera, one of the main species of tea oil Camellia, shows LSI, in which SI occurs in the style, and pollen tube growth in the ovary ceases at 96 h after selfing. The CdS-RNase gene of Camellia drupifera was successfully cloned, and the gene showed a length of 922 bp and encoded 238 amino acids. CdS-RNase had typical S-RNase characteristics. The functional validation results revealed that CdS-RNase was a key female factor for SI in C. drupifera, indicating that S-RNase plays an important role in the plant LSI mechanism. CdS-RNase localized to the cell membrane, was specifically expressed in the style and transferred to the ovary, and inhibited pollen tube growth in the style and ovary. There was an "enhancer factor" for the expression of CdS-RNase in selfing styles. Between 24 h and 48 h after selfing, CdS-RNase expression was significantly upregulated 28.3 times, which was more than 2.5 times higher than the outcross expression level and more than 5 times lower than the cross expression level. A total of 28 CdS-RNase proteins were identified, including 6 CdSm-RNases, and outcrossing among different CdS-RNase genotypes had a significant effect on the fruit setting rate. It may be that mismatches among CdS-RNase genotypes and self-inbreeding are the main reasons for the low yield of C. drupifera. These results provide a basis for the systematic analysis of the LSI mechanism and for solving the SI problem of tea oil Camellia.

Effects of species and varietal differences on intergeneric cross-compatibility between apple and pear

Hybridization across species and genus has been utilized to improve agriculturally important traits in crops. These interspecific and intergeneric hybridizations are often inhibited by reproductive barriers at the prezygotic and postzygotic stages. In this study, we used intergeneric hybridization between apple and pear to investigate the effects of species and varietal differences on pollen tube growth, seed germination, and seedling survival in a wide range of germplasms. In apple pistils pollinated by pears, inferior pollen tube growth was often observed for European pear cultivars, whereas hybrid seedlings derived from a cross between apple and European pear showed higher survival rate. In contrast, Japanese pear and Chinese pear pollination resulted in higher fruit setting which likely resulted from a higher rate of compatible pistils but a lower rate of seedling survival. Our results suggest the presence of both prezygotic and postzygotic barriers that occur independently in different crossing combinations. We also developed a Cleaved Amplified Polymorphic Sequence marker for detecting intergeneric hybrids derived from a wide range of crosses between apples and pears. The results of the present study will help in the development of intergeneric hybrids between apple and pear for introducing valuable traits from a diverse gene pool.

Reproductive biology of the "Brazilian pine" (Araucaria angustifolia-Araucariaceae): the pollen tube growth and the seed cone development.

In Araucaria angustifolia, the seed scale is part of the ovule, the female gametophyte presents a monosporic origin and arises from a coenocytic tetrad, and the pollen tube presents a single axis. The seed cone of conifers has many informative features, and its ontogenetic data may help interpret relationships among function, development patterns, and homology among seed plants. We reported the seed cone development, from pollination to pre-fertilization, including seed scale, ovule ontogeny, and pollen tube growth in Araucaria angustifolia. The study was performed using light microscopy, scanning electron microscopy, and X-ray microcomputed tomography (μCT). During the pollination period, the ovule arises right after the seed scale has emerged. From that event to the pre-fertilization period takes about 14months. Megasporogenesis occurs three weeks after ovule formation, producing a coenocytic tetrad. At the same time as the female gametophyte's first nuclear division begins, the pollen tube grows through the seed scale adaxial face. Until maturity, the megagametophyte goes through the free nuclei stage, cellularization stage, and cellular growth stage. Along its development, many pollen tubes develop in the nucellar tissue extending straight toward the female gametophyte. Our observations show that the seed scale came out of the same primordia of the ovule, agreeing with past studies that this structure is part of the ovule itself. The formation of a female gametophyte with a monosporic origin that arises from a coenocytic tetrad was described for the first time in conifers, and the three-dimensional reconstruction of the ovule revealed the presence of pollen tubes with only one axis and no branches, highlighting a new pattern of pollen tube growth in Araucariaceae.

Influence of pollination treatments and temperature regimes on progamic phase and fruit set in three European plum (Prunus domestica L.) cultivars tollerant/resistant to Sharka virus

Pollination and temperature are predominant factors affecting the progamic phase of sexual plant reproduction. In light of climate change and the emergence of increasing temperatures, new knowledge is necessary to encourage fruit breeding programmes to develop cultivars adapted to these changes, as well as to find the best incompatibility relationships with the aim of achieving high fruit quality and yield. This research was conducted to reveal the effect of pollination treatments and temperature on the progamic phase and fruit set in three European plum (Prunus domestica L.) cultivars (‘Jojo’, ‘Hanita’ and ‘Katinka’) resistant/tolerant to Sharka virus using fluorescent microscopy. Reproductive parameters (flowering overlapping, pollen germination, number of pollen tubes in the upper part of the style and locule of the ovary, dynamics of pollen tubes growth and initial and final fruit set) were investigated in vivo under different pollination treatments (cross, open and self-pollination). The influence of different temperature regimes (20 °C, 23 °C and 26 °C) on the progamic phase was also observed under cross and self-pollination. The pollination treatments significantly influenced the number of pollen tubes in the style and ovary, as well as dynamics of pollen tubes in different regions of pistil. Pollen tubes showed better growth in different crossing combinations compared to open and self-pollination. Higher temperatures accelerated pollen tube growth and reduced the time they need to reach ovules. Generally, ‘Jojo’ appeared to be the best pollenizer among the examined cultivars, while ‘Katinka’ exhibited the best response of pollen tube growth to various temperature regimes. The differences observed between growth of the pollen tubes in each genotype-temperature combination revealed that certain crossing combinations were better adapted to higher temperatures so this can be taken into account as one of the adjustments to climate change. The ability of cultivars to respond to high temperatures in the progamic phase can also be helpful for breeding programs in developing new cultivars better adapted to different temperature conditions.

Unilateral cross-incompatibility between Camellia oleifera and C. yuhsienensis provides new insights for hybridization in Camellia spp.

Camellia yuhsienensis was used to cross with Camellia oleifera to improve the resistance of oil camellia anthracnose. However, unilateral cross-incompatibility (UCI) between C. oleifera and C. yuhsienensis was found during the breeding process. Five C.oleifera cultivars and four C. uhsienensis materials were tested to confirm the UCI between C. oleifera and C. yuhsienensis. 'Huashuo' (HS) and 'Youza 2' (YZ2) were used to represent these two species to characterize the UCI, including pollen tube growth, fertilization and fruit development. The results demonstrated that UCI was prevalent between C. oleifera and C. yuhsienensis. The asynchronous flowering period was a pre-pollination barrier that limited mating between these two species under natural conditions. Interspecific pollen tubes were observed through the styles of these two plants, though the growth rates differed considerably. At 96 hours after pollination, the pollen tube of YZ2 barely entered the ovule, but remained at the base of the style and became swollen. However, the HS pollen tube entered the ovule 48 hours after pollination, double fertilization was observed, and the fruit and seeds developed commonly. Relative to compatible combinations, most unfertilized ovules in incompatible combinations failed to grow, turned brown 150 days after pollination, and the fruits were smaller than expected with uneven enlargement. Investigations on both semi-in vivo and in vitro pollen tubes gave us new idea for thought: the HS style has a stronger inhibitory effect on the interspecific pollen tubes, while calcium alleviates the inhibitory of styles but failed to prevent the appearance of abnormal pollen tube morphology. This study provides useful information on interspecific hybridization between C. oleifera and C. yuhsienensis for understanding reproductive isolation mechanisms and breeding programs in genus Camellia.

Movement of Lipid Droplets in the Arabidopsis Pollen Tube Is Dependent on the Actomyosin System.

The growth of pollen tubes, which depends on actin filaments, is pivotal for plant reproduction. Pharmacological experiments showed that while oryzalin and brefeldin A treatments had no significant effect on the lipid droplets (LDs) trafficking, while 2,3-butanedione monoxime (BDM), latrunculin B, SMIFH2, and cytochalasin D treatments slowed down LDs trafficking, in such a manner that only residual wobbling was observed, suggesting that trafficking of LDs in pollen tube is related to F-actin. While the trafficking of LDs in the wild-type pollen tubes and in myo11-2, myo11b1-1, myo11c1-1, and myo11c2-1 single mutants and myo11a1-1/myo11a2-1 double mutant were normal, their trafficking slowed down in a myosin-XI double knockout (myo11c1-1/myo11c2-1) mutant. These observations suggest that Myo11C1 and Myo11C2 motors are involved in LDs movement in pollen tubes, and they share functional redundancy. Hence, LDs movement in Arabidopsis pollen tubes relies on the actomyosin system.

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia. Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells' major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support. This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at: https://github.com/badain/amebas.git

How pollen tubes fight for food: the impact of sucrose carriers and invertases of Arabidopsis thaliana on pollen development and pollen tube growth.

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.