The estimation of pollen viability in rice

Methods for testing pollen viability in rice were evaluatedby comparing staining with aniline blue in lactophenol, twotetrazolium salts and fluorescein diacetate with germinationin an in vitro culture medium. Staining with thiazolyl blue(MTT) was nicely correlated with germination. Under non-saline conditions, rice pollen was only viable fora short period of time, with an approximately 50% loss of viabilitywithin 20 min of shedding. When plants were salinized at eitherpanicle initiation or the booting stage, pollen viability wasreduced. Key words: Rice, Oryza sativa, pollen viability, salinity     

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.     

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.     

Actin and pollen tube growth

Summary Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen tube growth.     

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.     

Signalling pathways in pollen germination and tube growth

Summary. Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited. Correspondence and reprints: Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

Resilience of rice (Oryza spp.) pollen germination and tube growth to temperature stress

Resilience of rice cropping systems to potential global climate change will partly depend on the temperature tolerance of pollen germination (PG) and tube growth (PTG). Pollen germination of high temperature‐susceptible Oryza glaberrima Steud. (cv. CG14) and Oryza sativa L. ssp. indica (cv. IR64) and high temperature‐tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6–45.4 °C temperature gradient system. Mean maximum PG was 85% at 27 °C with 1488 μm PTG at 25 °C. The hypothesis that in each pollen grain, the minimum temperature requirements (T_n) and maximum temperature limits (T_x) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (T_x(50)) was the lowest (29.8 °C) in IR64 compared with CG14 (34.3 °C) and N22 (35.6 °C). Standard deviation (s_x) of T_x was also low in IR64 (2.3 °C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1 mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters T_x(50) and s_x in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.     

Pistil strategies controlling pollen tube growth

The progamic phase appears especially well suited for pollen-pistil interaction. During this phase the pistil supports pollen germination and tube growth, and provides an adequate environment, nutrition and directional cues. However, this support does not occur indiscriminantly and some mechanisms operating in the pistil constrain pollen tube growth. An active, regulated constraint is the self-incompatibility reaction, but moderate restrictions of pollen tube growth also occur in compatible matings. These moderate restrictions involve reduced support by the pistil and they operate through two main strategies; one is by decreasing the amount of support and the other is by varying the time at which this support is provided. In this minireview, we examine the evidence that is accumulating for both support and constraint of pollen tube growth by the pistil and discuss the benefits of this dual system.     

Effect of toluidine blue on pollen germination and pollen tube growth

Toluidine blue is known to induce gynogenic haploids in significant numbersin Populus]. Because the efficacy of a chemical in inducing gynogenesis depends largely on its effeot on pollen germination, on pollen tube growth, and on male gamete formation, the effect of toluidine blue (0, 1, 10 and 100 mgl-1) on these processes was studied in treated pistils ofSolatium nigrum (4 X), as well as on cultured pollen grains ofS. nigrum andTrigonella foenumgraecum. Irrespective of the time of application, toluidine blue (1 and 10 mg I-1) had no effect on pollen germination or pollen tube growth in pistils ofS. nigrum; at 100 mg I-1 it invariably inhibited both the processes. Almost similar responses were elicited by cultured pollen grains. InT. foenum-graecum toluidine blue had no effeot on pollen germination and suppressed tube growth. Gamete formation was inhibited, to various degrees, at all the concentrations tested; at 100 ing I-1 hardly any pollen tube showed gamete formation. Based on our results, and those on other systems, the potentiality of toluidine blue as an inducer of gynogenesis has been analysed.     

Nucellar beak structure and pollen tube growth in Cucurbitaceae

The nucellar beak is a proboscis-like outgrowth of the nucellus at the micropylar end, being the obligatory path for the pollen tube entering the ovule. Among the few angiosperm families with nucellar beak, Cucurbitaceae is remarkable because the pollen tube may develop at least two types of growth within the nucellar beak: tubular and ampulliform. Wondering about the possibility that Cucurbitaceae ovules may express some histological variation that could be related to pollen tube growth within the nucellar beak, we performed a compared anatomical and histochemical study of the nucellar beak and the pollen tube growth of ten species of Cucurbitaceae. Results show that Cucurbitaceae ovules are diverse in size and proportions (of integuments, nucellar body, and nucellar beak), and they have at least four types of nucellar beak histology: pectic-tracked, secretory-like, amylaceous, and mixed. Amylaceous and mixed nucellar beaks are related to the ampulliform growth of the pollen tube, which could have appeared independently in most derived tribes of Cucurbitaceae, although information about nucellar beak structure in the basal tribes is still needed. In addition, the understanding of the relation between amylaceous nucellar beaks and the ampulliform growth of the pollen tube, whose function is still to be discovered, might open the possibility of a unique model of pollen tube-ovule co-evolution in angiosperms.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

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.     

花粉管生长调控的研究进展

本文从花粉管的生长特性、细胞质组成、细胞骨架、细胞壁的结构与合成、Ｃａ２＋通道和向性生长机制六个方面,综述了近些年来对花粉管生长调控研究的进展。     

Pollen-specific pectin methylesterase involved in pollen tube growth

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.