Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1‐3 pskr2‐1 and tpst‐1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1‐3 pskr2‐1 and of tpst‐1 were shorter. In planta, growth of wild‐type pollen in pskr1‐3 pskr2‐1 and tpst‐1 flowers appeared slower than growth in wild‐type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1‐3 pskr2‐1 and in tpst‐1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross‐pollination experiments with wild‐type, pskr1‐3 pskr2‐1 and tpst‐1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.     

Gametophytic pollen tube guidance

The concept of a pollen tube attractant was proposed in the late nineteenth century when pollen tubes were found to grow toward excised pistil tissues on medium. Since then, for about 140 years, plant biologists have tried to identify the pollen tube attractants. However, no molecule has been convincingly demonstrated to be the true attractant that actually controls the navigation of pollen tubes in the pistil. The past decade has seen substantial progress in this field in terms of our understanding of the various mechanisms of pollen tube guidance. It was suggested that diffusible pollen tube attractants might provide localized signals that affect the directional growth of the pollen tube, especially in the last phase of guidance by the target female gametophyte. Here, we review the mechanisms of pollen tube guidance, with special focus on the gametophytic guidance and the attractant. The necessary and appropriate conditions required by the true attractant will be discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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

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

AMINOPHOSPHOLIPID ATPASE3 guides pollen tubes by regulating the distribution of anionic phospholipids to affect the precise localization of certain pollen-specific receptor kinases at pollen tubes.

IN A NUTSHELL Background: In flowering plants, pollen tube guidance regulates the rapid growth and timely targeting of the pollen tube to the ovule in the pistil during sexual reproduction, when signaling between the male and female gametophytes occur. The small peptide-RLK signaling module is essential for the interaction between the male and female gametophyte. Certain members of the pollen-specific receptor kinase (PRK) family have different subcellular localization patterns in Arabidopsis pollen tubes and play critical roles in pollen tube growth and guidance. However, the molecular mechanisms that regulate and maintain the polar localization of PRKs at the pollen tube tip are still unknown. Question: We were interested in exploring how Arabidopsis P4-ATPase (aminophospholipid ATPase, ALA) precisely regulates pollen tube guidance and maintains the polar localization patterns of PRK6 and PRK3. How plant ALA family members regulate pollen tube guidance has not yet been documented. Findings: The loss of ALA3 function not only caused sluggish pollen tube growth and aberrant ovule targeting but also affected the polar localization patterns of several PRKs at the pollen tube tip. Members of the PRKs family can directly interact with anionic phospholipids such as phosphatidylserine (PS), and the capacity of PRK3/6 to bind anionic phospholipids is crucial for both their polar localization and physiological functions. ALA3 establishes and maintains the polar distribution of PS, which influences secretory vesicles-mediated polar trafficking at the pollen tube tip to affect the distribution of PRK3 and PRK6. On the other hand, PS might also directly recruit PRK3 and PRK6 to the pollen tube tip and sustain their localization. Next steps: The localization of PRKs is a complex, finely regulated process. The C-termini of PRKs may also affect their polar distribution. More research is required to reveal how the C-terminus domain precisely controls the localization of PRKs.     

POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis

The pollen tube germinates from pollen and, during its migration, it perceives and responds to guidance cues from maternal tissue and from the female gametophyte. The putative female cues have recently been identified, but how the pollen tube responds to these signals remains to be unveiled. In a genetic screen for male determinants of the pollen tube response, we identified the pollen defective in guidance1 (pod1) mutant, in which the pollen tubes fail to target the female gametophyte. POD1 encodes a conserved protein of unknown function and is essential for positioning and orienting the cell division plane during early embryo development. Here, we demonstrate that POD1 is an endoplasmic reticulum (ER) luminal protein involved in ER protein retention. Further analysis shows that POD1 interacts with the Ca(2+) binding ER chaperone CALRETICULIN3 (CRT3), a protein in charge of folding of membrane receptors. We propose that POD1 modulates the activity of CRT3 or other ER resident factors to control the folding of proteins, such as membrane proteins in the ER. By this mechanism, POD1 may regulate the pollen tube response to signals from the female tissues during pollen tube guidance and early embryo patterning in Arabidopsis thaliana.     

MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.     

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3′UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.     

A Brassica napus skp1-like gene promoter drives GUS expression in Arabidopsis thaliana male and female gametophytes

We isolated a gene, BnSKP1γ1, expressed in rapeseed (Brassica napus) microspores, which encodes a protein closely related to the Saccharomyces cerevisiae Skp1p protein previously shown to play a role in cell cycle regulation. Twelve SKP1-related genes have already been identified in the Arabidopsis thaliana genome. Using a PCR-based strategy, we isolated three other genes. To date, most data available concerning the function of the SKP1-related genes in plants are indirect. Studies on transgenic A. thaliana plants showthat a 1100-bp BnSKP1γ1 promoter fragment can direct GUS expression in female gametophytes soon after the first haploid mitosis and in male gametophytes from the tetrade stage. No GUS expression can be detected in sporophytic tissues. RT-PCR experiments suggest that this gene is expressed in a similar way in rapeseed. This is the first reported case of a gene exhibiting such an expression pattern in angiosperms. Received: 5 October 1999 / Revision accepted: 28 March 2000     

The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana, named feronia after the Etruscan goddess of fertility, which impairs this process. In the feronia mutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.     

The DC1‐domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain‐containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine‐ and histidine‐rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre‐vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 – a member of the SNARE vesicle‐associated protein family and previously related to a sterol‐binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.     

Metabolism and roles of phosphatidylinositol 3-phosphate in pollen development and pollen tube growth in Arabidopsis

Phosphoinositides play important roles in eukaryotic cells, although they constitute a minor fraction of total cellular lipids. Specific kinases and phosphatases function on the regulation of phosphoinositide levels. Phosphatidylinositol 3-phosphate (PtdIns3P), a molecule of phosphoinositides regulates multiple aspects of plant growth and development. In this article, we introduce and discuss the kinases and phosphatases involved in PtdIns3P metabolism and their roles in pollen development and pollen tube growth in Arabidopsis.     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.