Autotetraploidy in Cucumis zeyheri and a derived new species C. diniae (Cucurbitaceae)

Three cytotypes at the diploid and tetraploid level of Cucumis zeyheri were investigated by means of multivariate morphological analysis, for pollen structure and by analysis of meiosis. Two main groups emerged after principal component and cluster analyses. Diploids and autotetraploids were placed in one group designated as C. zeyheri , the allotetraploids formed a separate group. Autotetraploids showed a mean number of one quadrivalent per pollen mother cell, together with 10% tetra-porate pollen grains indicating an autoploid origin. The allotetraploids may have originated from polyploidization after hybridization of C. zeyheri and C. prophetarum. They are described as the new species C. diniae , which differs from C. zeyheri in leaf colour and width of leaf lobes, and from C. prophetarum in the concolour fruits. Leaf shape and leaf incision is intermediate between the parents.     

Pathways to polyploidy: indications of a female triploid bridge in the alpine species <Emphasis Type="Italic">Ranunculus kuepferi</Emphasis> (Ranunculaceae)

Polyploidy is one of the most important evolutionary processes in plants. In natural populations, polyploids usually emerge from unreduced gametes which either fuse with reduced ones, resulting in triploid offspring (triploid bridge), or with other unreduced gametes, resulting in tetraploid embryos. The frequencies of these two pathways, and male versus female gamete contributions, however, are largely unexplored. Ranunculus kuepferi occurs with diploid, triploid and autotetraploid cytotypes in the Alps, whereby diploids are mostly sexual, while tetraploids are facultative apomicts. To test for the occurrence of polyploidization events by triploid bridge, we investigated 551 plants of natural populations via flow cytometric seed screening. We assessed ploidy shifts in the embryo to reconstruct female versus male gamete contributions to polyploid embryo and/or endosperm formation. Seed formation via unreduced egg cells (B_III hybrids) occurred in all three cytotypes, while only in one case both gametes were unreduced. Polyploids further formed seeds with reduced, unfertilized egg cells (polyhaploids and aneuploids). Pollen was highly variable in diameter, but only pollen >27 μm was viable, whereby diploids produced higher proportions of well-developed pollen. Pollen size was not informative for the formation of unreduced pollen. These results suggest that a female triploid bridge via unreduced egg cells is the major pathway toward polyploidization in R. kuepferi, maybe as a consequence of constraints of endosperm development. Triploids resulting from unreduced male gametes were not observed, which explains the lack of obligate sexual tetraploid individuals and populations. Unreduced egg cell formation in diploids represents the first step toward apomixis.     

HEAT INJURY DURING FLORAL DEVELOPMENT IN COWPEA (VIGNA UNGUICULATA,FABACEAE)

High night temperatures during floral development induce male sterility in cowpea (Vigna unguiculata [L.] Walp.). The objectives of this study were to determine: the possible causes of the male sterility; the stage of floral development when damage due to heat stress occurs; and whether specific tissues are damaged during the period of sensitivity to heat. Plants were grown under controlled temperatures in both greenhouses and growth chambers in separate experiments. Floral development was normal under a night temperature of 20 C, whereas flowers developed under high night temperature (30 C) set no pods due to low pollen viability and anther indehiscence. Anthers developed under 33/30 C day/night temperatures did not exhibit endothecial formation, whereas anthers developed under 33/20 C day/night temperatures exhibited normal development of the endothecial layer. Reciprocal transfers of plants between chambers with high or optimum night temperature demonstrated that the stage of floral development most sensitive to heat stress occurs 9 to 7 d before anthesis. Anthers developed under either optimal or high night temperatures were compared cytologically. Development was similar through meiosis, but after tetrad release, which occurred 8 d before anthesis, the tapetal layer degenerated prematurely under high night temperature. Premature degeneration of the tapetal layer and lack of endothecial development may be responsible for the low pollen viability, low anther dehiscence, and low pod set under high night temperatures.     

Molecular Cytogenetic Analysis of Polyploidization in the Anther Tapetum of Diploid and Autotetraploid Arabidopsis thaliana Plants

Like those of most angiosperms, vegetative tissues of Arabidopsisthaliana undergo high levels of endopolyploidization. One suchtissue is the anther tapetum which plays a role in male sporo-and gametogenesis. The degree of polyploidization of the tapetumvaries from species to species. Although the role of this processis not yet fully understood, it may be linked to functioningof the tapetum, increasing the copy number of genes needed forthe synthesis of specific factors required by developing pollenmother cells (PMCs) and pollen grains. The present study focusedon polyploidization during the development of the tapetum ofArabidopsis thaliana. The aim was to outline the mode of tapetumpolyploidization in this model plant species and to establishan efficient method for analysing ploidy levels in differentiatedcells. The course and degree of tapetum polyploidization inArabidopsis was analysed in interphase nuclei using fluorescencein situ hybridization (FISH) with repetitive DNA (45S rDNA).The stages of development of the tapetum were analysed alongsidemeiosis in PMCs. The majority of tapetal cells undergo two,maximally three, rounds of divisions. Tapetal nuclei have usuallydivided by metaphase I of meiosis of PMCs. The pattern of tapetumpolyploidization was similar in diploid and autotetraploid plantsand is thus not affected by increasing amounts of maternal plantDNA. The tapetum of autotetraploid plants exhibits a higherfrequency of additional division than seen in diploid plants.Copyright 2001 Annals of Botany Company Arabidopsis thaliana, autotetraploid, FISH, rDNA polyploidization, tapetum     

Meiotic mutants of Medicago sativa show altered levels of alpha- and beta-tubulin.

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).     

High temperature limits in vivo pollen tube growth rates by altering diurnal carbohydrate balance in field-grown Gossypium hirsutum pistils

It has recently been reported that high temperature slows in vivo pollen tube growth rates in Gossypium hirsutum pistils under field conditions. Although numerous physical and biochemical pollen-pistil interactions are necessary for in vivo pollen tube growth to occur, studies investigating the influence of heat-induced changes in pistil biochemistry on in vivo pollen tube growth rates are lacking. We hypothesized that high temperature would alter diurnal pistil biochemistry and that pollen tube growth rates would be dependent upon the soluble carbohydrate content of the pistil during pollen tube growth. G. hirsutum seeds were sown on different dates to obtain flowers exposed to contrasting ambient temperatures but at the same developmental stage. Diurnal pistil measurements included carbohydrate balance, glutathione reductase (GR; EC 1.8.1.7), soluble protein, superoxide dismutase (SOD; EC 1.15.1.1), NADPH oxidase (NOX; EC 1.6.3.1), adenosine triphosphate (ATP), and water-soluble calcium. Soluble carbohydrate levels in cotton pistils were as much as 67.5% lower under high temperature conditions (34.6 °C maximum air temperature; August 4, 2009) than under cooler conditions (29.9 °C maximum air temperature; August 14, 2009). Regression analysis revealed that pollen tube growth rates were highly correlated with the soluble carbohydrate content of the pistil during pollen tube growth (r² = 0.932). Higher ambient temperature conditions on August 4 increased GR activity in the pistil only during periods not associated with in vivo pollen tube growth; pistil protein content declined earlier in the day under high temperatures; SOD and NOX were unaffected by either sample date or time of day; pistil ATP and water soluble calcium were unaffected by the warmer temperatures. We conclude that moderate heat stress significantly alters diurnal carbohydrate balance in the pistil and suggest that pollen tube growth rate through the style may be limited by soluble carbohydrate supply in the pistil.     

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.     

Effects of Temperature on Pollen Viability in Mango cv. 'Kensington'

The response of pollen development to low or high temperatureregimes was studied to determine the conditions suitable forthe formation of fertile pollen in the mango cv. 'Kensington'.The phase most sensitive to the degree and duration of temperaturestress was that from meiosis to the pre-vacuolate microspore(about 3 d duration at 25/20 °C) though vacuolated microsporeswere also sensitive to low temperature. Night temperatures below10 °C resulted in pollen grains with a low viability (<50%). A temperature between 15 and 33 °C during the phasefrom meiosis to the pre-vacuolate microspore was optimum forpollen development (70-85% pollen viability). Analysis of field records showed a linear negative correlationbetween percentage of pollen viability and number of days whichhad a mean night temperature lower than 10 °C during theperiod from meiosis to early mature stage (y = 77·7-3·4x,r² = 0·60). The temperature sensitive phase was estimatedto begin 155 degree days D = [(T_max + T_min)/2 - 10] before anthesisand to end 78 degree days before anthesis. This equation maybe useful as a means of predicting pollen viability in the fieldfrom temperature records and thus fruit set, date of maturityand yield. It may also aid in the selection of areas for growingmangoes in marginal climates.Copyright 1994, 1999 Academic Press Mangifera indica L. mango, microsporogenesis, pollen development, viability, sterility, temperature     

The effect of high temperature and high atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination

Pollen viability and germination are known to be sensitive to high temperature (HT). However, the mode by which high temperature impairs pollen functioning is not yet clear. In the present study, we investigated the effect of high temperature on changes occurring in carbohydrate of bell pepper (Capsicum annuum L. cv. Mazurka) pollen in order to find possible relations between these changes and pollen germination under heat stress. When pepper plants were maintained under a moderate HT regime (32/26 degrees C, day/night) for 8 days before flowers have reached anthesis, pollen count at anthesis was similar to that found in plants grown under normal temperatures (NT 28/22 degrees C). However, the in vitro germination, carried out at 25 degrees C, of pollen from HT plants was greatly reduced. This effect matched the marked reduction in the number of seeds per fruit in the HT plants. Maintaining the plants at high air CO2 concentration (800 &mgr;mol mol-1 air) in both temperature treatments did not affect the in vitro germination of pollen from NT plants, but restored germination to near the normal level in pollen from HT plants. Under NT conditions, starch, which was negligible in pollen at meiosis (8 days before anthesis, A-8) started to accumulate at A-4 and continued to accumulate until A-2. From that stage until anthesis, starch was rapidly degraded. On the other hand, sucrose concentration rose from stage A-4 until anthesis. Acid invertase (EC 3.2.1.26) activity rose parallel with the increase of sucrose. In pollen from HT plants, sucrose and starch concentrations were significantly higher at A-1 pollen than in that of NT plants. Under high CO2 conditions, the sucrose concentration in the pollen of HT plants was reduced to levels similar to those in NT pollen. In accordance with the higher sucrose concentration in HT pollen, the acid invertase activity in these pollen grains was lower than in NT pollen. The results suggest that the higher concentrations of sucrose and starch in the pollen grains of HT plants may result from reduction in their metabolism under heat stress. Elevated CO2 concentration, presumably by increasing assimilate availability to the pollen grain, may alleviate the inhibition of sucrose and starch metabolism, thereby increasing their utilization for pollen germination under the HT stress. Acid invertase may have a regulatory role in this system.     

Polyploidy‐associated genomic instability in Arabidopsis thaliana

Formation of polyploid organisms by fertilization of unreduced gametes in meiotic mutants is believed to be a common phenomenon in species evolution. However, not well understood is how species in nature generally exist as haploid and diploid organisms in a long evolutionary time while polyploidization must have repeatedly occurred via meiotic mutations. Here, we show that the ploidy increased for two consecutive generations due to unreduced but viable gametes in the Arabidopsis cyclin a1;2‐2 (also named tardy asynchronous meiosis‐2) mutant, but the resultant octaploid plants produced progeny of either the same or reduced ploidy via genomic reductions during meiosis and pollen mitosis. Ploidy reductions through sexual reproduction were also observed in independently generated artificial octaploid and hexaploid Arabidopsis plants. These results demonstrate that octaploid is likely the maximal ploidy produced through sexual reproduction in Arabidopsis. The polyploidy‐associated genomic instability may be a general phenomenon that constrains ploidy levels in species evolution. genesis 48:254–263, 2010. © 2010 Wiley‐Liss, Inc.     

A CYTOGENETIC ANALYSIS OF CERTAIN POLYPLOIDS IN GRINDELIA (COMPOSITAE)

Two diploid taxa, Grindelia procera and G. camporum, and 3 tetraploid ones, G. camporum, G. hirsutula, and G. stricta, have been studied to ascertain their interrelationships. Meiosis in diploid parental strains was regular, the common chromosome configuration being 5 rod bivalents and 1 ring bivalent. The average chiasmata frequency per chromosome was 0.60. Pollen fertility was about 90% in all strains examined. Diploid interspecific hybrids had normal meiosis with an average chiasmata frequency of 0.56 per chromosome. No heterozygosity for inversions or interchanges was detected, and pollen fertility was above 85%. Meiosis in parental tetraploid strains was characterized by the presence of quadrivalents in addition to a complementary number of bivalents. The average chiasmata frequency per chromosome was 0.59 and pollen fertility was generally about 80%. Tetraploid interspecific hybrids also had quadrivalents, normal meiosis, and high pollen fertility. Close genetic relationships between the diploids and between the tetraploids are indicated, and geographical, ecological, and seasonal barriers to gene exchange exist. Attempts to obtain hybrids between diploids and tetraploids were successful in a few cases. The hybrids were tetraploid and had normal meiosis and fertility similar to parental and F₁ tetraploids. Their origin was by the union of unreduced gametes of the diploid female parent and normal pollen from the tetraploid parent. On the basis of chromosome homology, normal meiosis, plus high fertility exhibited in the diploid, tetraploid, and diploid X tetraploid interspecific hybrids, these species of Grindelia are considered to be a part of an autopolyploid complex. Gene exchange between diploids and diploids, tetraploids and tetraploids, and diploids and tetraploids is possible. Tetraploid G. camporum may have originated by hybridization between G. procera and diploid G. camporum with subsequent doubling of chromosomes and selection for the combined characteristics of the diploids.     

Genome duplication effects on pollen development and the interrelated physiological substances in tetraploid rice with polyploid meiosis stability

The breeding of polyploid rice made no breakthrough for a long time because of low seed set. The discovery and application of polyploid meiosis stability (PMeS) material played a pivotal role in solving this problem. Our results indicated that genome duplication led to different outcomes in different rice cultivars in terms of pollen fertility, viability, and the accumulation of important physiological substances such as free proline and endogenous hormones. Pollen from the PMeS HN2026-4X lines showed a high fertility and viability similar to those of HN2026-2X (4X indicates tetraploid while 2X indicates the diploid), whereas both rates decreased dramatically in Balilla-4X. The results of pollen microstructure and ultrastructure investigations suggested that the pollen development pattern in HN2026-4X appeared normal at all stages, but a lot of changes were discovered in Balilla-4X. Stable meiosis, timely tapetum degradation, and normal mitochondria development were critical factors insuring the high frequency pollen fertility of PMeS rice. The free proline content increased markedly in HN2026-4X as compared to HN2026-2X, but it was decreased for Balilla-4X. Genome duplication effects on regulating endogenous hormones accumulation in pollen were evident, resulting in the clear difference between PMeS HN2026-4X and Balilla-4X. The accumulation of IAA, ZR, and GA in mature pollen distinguished HN2026-4X from Balilla-4X, which was linked to normal pollen development. In particular, the excessive accumulation of ABA at the meiosis stage may be correlated to disorganized meiosis in Balilla-4X. All the results provided unequivocal evidence that genome duplication played specific roles in the normal pollen development of PMeS HN2026-4X.     

Cytological studies on a haploid cultivar of pelargonium,and its colchicine-induced diploids

Kleine Liebling is a haploid of unusual interest; not only is it a monoploid, but at the same time a successful horticultural variety. Analysis made of pollen mother cell meiosis revealed a very low frequency of bivalents, and evidence of neocentric activity among the univalents. Colchicine-induced diploid plants were synthesised, but contrary to expectation, microsporogenesis was very erratic, and pairing never complete. As in the haploid from which they were derived, a high proportion of dyads formed the final product of meiosis. It was concluded that both these irregularities were the result of mutations effecting the genetic control of meiosis.     

Focus Issue on the Plant Physiology of Global Change: Production of Diploid Male Gametes in Arabidopsis by Cold-Induced Destabilization of Postmeiotic Radial Microtubule Arrays

Whole-genome duplication through the formation of diploid gametes is a major route for polyploidization, speciation, and diversification in plants. The prevalence of polyploids in adverse climates led us to hypothesize that abiotic stress conditions can induce or stimulate diploid gamete production. In this study, we show that short periods of cold stress induce the production of diploid and polyploid pollen in Arabidopsis (Arabidopsis thaliana). Using a combination of cytological and genetic analyses, we demonstrate that cold stress alters the formation of radial microtubule arrays at telophase II and consequently leads to defects in postmeiotic cytokinesis and cell wall formation. As a result, cold-stressed male meiosis generates triads, dyads, and monads that contain binuclear and polynuclear microspores. Fusion of nuclei in binuclear and polynuclear microspores occurs spontaneously before pollen mitosis I and eventually leads to the formation of diploid and polyploid pollen grains. Using segregation analyses, we also found that the majority of cold-induced dyads and triads are genetically equivalent to a second division restitution and produce diploid gametes that are highly homozygous. In a broader perspective, these findings offer insights into the fundamental mechanisms that regulate male gametogenesis in plants and demonstrate that their sensitivity to environmental stress has evolutionary significance and agronomic relevance in terms of polyploidization.The spontaneous formation of polyploid species through whole-genome duplication is a major force driving diversification and speciation in plant evolution (Wang et al., 2004). The redundant genomic material produced by polyploidization provides genotypic plasticity that facilitates adaptation and confers enhanced competitiveness compared with diploid progenitors (Adams and Wendel, 2005a, 2005b; Leitch and Leitch, 2008). Molecular analyses suggest that the genomes of most angiosperms (more than 90%) retain evidence of one or more ancient genome-wide duplication events (Cui et al., 2006). Moreover, recently, Wood et al. (2009) established that up to 15% of angiosperm and 31% of gymnosperm speciation events were accompanied by polyploidization. Polyploidization in plants is also commercially beneficial. Many important crop species including wheat (Triticum aestivum), potato (Solanum tuberosum), tobacco (Nicotiana tabacum), coffee (Coffea arabica), and numerous fruit varieties are polyploid (Bretagnolle and Thompson, 1995). Although several mechanisms can yield polyploids, it is thought that most polyploid plants are formed by the spontaneous production and fusion of diploid (2n) gametes (Bretagnolle and Thompson, 1995; Ramsey and Schemske, 1998). However, despite the evolutionary and agricultural significance of sexual polyploidization in plants (Ramanna and Jacobsen, 2003), the molecular mechanism underlying 2n gamete formation in natural populations is poorly understood.Several cytological defects lead to diploid gamete formation in both male and female reproductive lineages. In some species, premeiotic and postmeiotic genome doubling events are reported, but diploid gametes typically result from a defect in one of the two meiotic divisions, a phenomenon referred to as “restitution” (Bretagnolle and Thompson, 1995; Ramsey and Schemske, 1998). Meiotic restitution mechanisms are categorized into three classes: (1) omission of one of the meiotic cell divisions; (2) alterations in meiosis I (MI) or meiosis II (MII) spindle morphology; or (3) defects in meiotic cytokinesis (Ramanna and Jacobsen, 2003). Additionally, depending on the genetic makeup of the resulting 2n gametes, meiotic restitution mechanisms can be further subdivided into two classes: first division restitution (FDR) and second division restitution (SDR). In FDR, the sister chromatids disjoin and segregate to opposite poles, yielding 2n gametes that largely retain the heterozygosity of the parental plant. In SDR, sister chromatids do not disjoin in MII and segregate to the same pole, generating highly homozygous 2n gametes (Köhler et al., 2010).Several genes governing 2n gamete formation have been identified and characterized in potato, maize (Zea mays), and Arabidopsis (Arabidopsis thaliana; Consiglio et al., 2004; Brownfield and Köhler, 2011). Mutations in Arabidopsis DYAD/SWITCH1 and maize ARGONAUTE104 (AGO104) and AM1 induce a complete loss of MI and, consequently, convert the meiotic cell cycle into a mitotic one (Ravi et al., 2008; Pawlowski et al., 2009; Singh et al., 2011). Lesions in Arabidopsis OSD1/GIG1 and TAM/CYCA2;1, two proteins involved in progression of the meiotic cell cycle, cause a complete loss of MII, generating highly homozygous 2n gametes in both male and female meiosis (d’Erfurth et al., 2009, 2010). Spindle-based meiotic restitution mechanisms have been reported in both Arabidopsis jason and atps1 mutants and in the potato ps mutant, in which parallel, fused, and tripolar spindles in male MII lead to the formation of FDR 2n spores (Mok and Peloquin, 1975; d’Erfurth et al., 2008; De Storme and Geelen, 2011). Disruption of postmeiotic male cytokinesis, which is regulated by a mitogen-activated protein kinase (MAPK) kinase signaling pathway, also results in polyploid gametes. Mutations in TES/STUD/AtNACK2, MKK6/ANQ1, and MPK4, three main components of the cytokinetic MAPK signaling cascade, induce a complete loss of cytokinesis following male meiosis, generating fully restituted tetraploid pollen grains (Hulskamp et al., 1997; Spielman et al., 1997; Soyano et al., 2003; Zeng et al., 2011).Despite progress on understanding cytological mechanisms and genetic factors governing the formation of 2n gametes in natural populations, less is known about the environmental factors involved. There is evidence that 2n gamete production can be stimulated by both biotic and abiotic stresses, such as nutritional deprivation, wounding, disease, herbivory, and temperature stress (Ramsey and Schemske, 1998). In Lotus tenuis, temperature stresses, and in particular high temperatures, increase the level of parallel spindle-driven 2n gamete production (Negri and Lemmi, 1998). Similarly, in rose (Rosa spp.), short periods of high temperature (48 h at 30°C–36°C) can induce cytomixis and parallel and tripolar spindles at male metaphase II, generating dyads and triads at the end of male sporogenesis (Pécrix et al., 2011). Low-temperature environments can also stimulate 2n gamete formation. For example, Solanum phureja grown in cool field environments produces more restituted spores compared with lines grown under normal conditions (McHale, 1983). Similarly, in Datura spp. and Achillea borealis, unreduced pollen formation is higher at low temperatures (Ramsey and Schemske, 1998; Ramsey, 2007). Recently, Mason et al. (2011) demonstrated that cold stress significantly stimulates 2n pollen production in some interspecific Brassica spp. hybrids. Temperature-induced diploid gamete formation is not restricted to plants. Low temperatures have also been shown to stimulate the formation of 2n spores in some animal species, particularly among fish and amphibians (Bogart et al., 1989; Mable et al., 2011). Moreover, ecological population studies have demonstrated that polyploid plant and animal species occur more frequently at higher altitudes and at latitudes closer to the poles (Beaton and Hebert, 1988; Barata et al., 1996; Dufresne and Hebert, 1998), leading to the suggestion that cold climates stimulate the production of polyploid gametes.In this study, we demonstrate that short periods of cold stress induce a development-specific production of meiotically restituted spores in Arabidopsis, which thereby constitutes an ideal model system to identify potential cytological and molecular factors involved in stress-induced sexual polyploidization. Using a combination of cytological and genetic approaches, we reveal the cytological basis for cold-induced meiotic restitution and additionally demonstrate that restituted binuclear and polynuclear spores spontaneously develop into diploid and polyploid pollen grains. We also use pollen tetrad-based segregation analysis to monitor the genetic makeup of cold-induced 2n gametes and Arabidopsis mutants to examine the potential role of some candidate regulators (e.g. TAM/CYCA1;2 and MKK2) in the sensitivity of male meiosis to low-temperature stress.     

Pollen vacuoles and their significance

Vacuoles of several types can be observed in pollen throughout its development. Their physiological significance reflects the complexity of the biological process leading to functional pollen grains. Vacuolisation always occurs during pollen development but when ripe pollen is shed the extensive translucent vacuoles present in the vegetative parts in previous stages are absent. Vacuole functions vary according to developmental stage but in ripe pollen they are mainly storage sites for reserves. Vacuoles cause pollen to increase in size by water accumulation and therefore confer some degree of resistance to water stress. Modalities of vacuolisation occur in pollen in the same manner as in other tissues. In most cases, autophagic vacuoles degrade organelles, as in the microspore after meiosis, and can be regarded as cytoplasm clean-up following the transition from the diploid sporophytic to the haploid gametophytic state. This also occurs in the generative cell but not in sperm cells. Finally, vacuoles have a function when microspores are used for pollen embryogenesis in biotechnology being targets for stress induction and afterwards contributing to cytoplasmic rearrangement in competent microspores.     

It is a matter of timing: asynchrony during pollen development and its consequences on pollen performance in angiosperms—a review

Functional pollen is needed to successfully complete fertilization. Pollen is formed inside the anthers following a specific sequence of developmental stages, from microsporocyte meiosis to pollen release, that concerns microsporocytes/microspores and anther wall tissues. The processes involved may not be synchronous within a flower, an anther, and even a microsporangium. Asynchrony has been barely analyzed, and its biological consequences have not been yet assessed. In this review, different processes of pollen development and lifetime, stressing on the possible consequences of their differential timing on pollen performance, are summarized. Development is usually synchronized until microsporocyte meiosis I (occasionally until meiosis II). Afterwards, a period of mostly asynchronous events extends up to anther opening as regards: (1) meiosis II (sometimes); (2) microspore vacuolization and later reduction of vacuoles; (3) amylogenesis, amylolysis, and carbohydrate inter-conversion; (4) the first haploid mitosis; and (5) intine formation. Asynchrony would promote metabolic differences among developing microspores and therefore physiologically heterogeneous pollen grains within a single microsporangium. Asynchrony would increase the effect of competition for resources during development and pollen tube growth and also for water during (re)hydration on the stigma. The differences generated by developmental asynchronies may have an adaptive role since more efficient pollen grains would be selected with regard to homeostasis, desiccation tolerance, resilience, speed of (re)hydration, and germination. The performance of each pollen grain which landed onto the stigma will be the result of a series of selective steps determined by its development, physiological state at maturity, and successive environmental constrains.     

Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen

Male reproductive development of rice (Oryza sativa L.) is very sensitive to drought. A brief, transitory episode of water stress during meiosis in pollen mother cells of rice grown under controlled environmental conditions induced pollen sterility. Anthers containing sterile pollen were smaller, thinner, and often deformed compared to normal anthers of well-watered plants. Only about 20% of the fully developed florets in stressed plants produced grains, compared to 90% in well-watered controls. Water stress treatments after meiosis were progressively less damaging. Levels of starch and sugars and activities of key enzymes involved in sucrose cleavage and starch synthesis were analyzed in anthers collected at various developmental stages from plants briefly stressed during meiosis and then re-watered. Normal starch accumulation during pollen development was strongly inhibited in stress-affected anthers. During the period of stress, both reducing and non-reducing sugars accumulated in anthers. After the relief of stress, reducing sugar levels fell somewhat below those in controls, but levels of non-reducing sugars remained higher than in controls. Activities of acid invertase and soluble starch synthase in stressed anthers were lower than in controls at comparable stages throughout development, during as well as after stress. Stress had no immediate effect on ADP-glucose pyrophosphorylase activity, but had an inhibitory aftereffect throughout post-stress development. Sucrose synthase activity, which was, relatively speaking, much lower than acid invertase activity, was only slightly suppressed by stress. The results show that it is unlikely that pollen sterility, or the attendant inhibition of starch accumulation, in water-stressed rice plants are caused by carbohydrate starvation per se. Instead, an impairment of enzymes of sugar metabolism and starch synthesis may be among the potential causes of this failure.     

Chilling to zero degrees disrupts pollen formation but not meiotic microtubule arrays in Triticum aestivum L.

Throughout the wheat‐growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat (Triticum aestivum L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments. Among the cultivars tested, the Australian cultivars Kite and Hartog had among the lowest levels of seed set due to chilling and their responses were investigated further. The developmental stage at exposure, the chilling temperature and length of exposure all influenced the level of sterility. The early period of booting, and specifically the +4 cm auricle distance class, was the most sensitive and corresponded to meiosis within the anthers. The response of microtubules to chilling during meiosis in Hartog was monitored, but there was little difference between chilled and control plants. Other abnormalities, such as plasmolysis and cytomixis increased in frequency, were associated with death of developing pollen cells, and could contribute to loss of fertility. The potential for an above‐zero chilling sensitivity in Australian spring wheat varieties could have implications for exploring the tolerance of wheat flower development to chilling and freezing conditions in the field.     

Cytological and morphology characteristics of natural microsporogenesis within Camellia oleifera

Camellia oleifera is believed to exhibit a complex intraspecific polyploidy phenomenon. Abnormal microsporogenesis can promote the formation of unreduced gametes in plants and lead to sexual polyploidy, so it is hypothesized that improper meiosis probably results in the formation of natural polyploidy in Camellia oleifera. In this study, based on the cytological observation of meiosis in pollen mother cells (PMCs), we found natural 2n pollen for the first time in Camellia oleifera, which may lead to the formation of natural polyploids by sexual polyploidization. Additionally, abnormal cytological behaviour during meiosis, including univalent chromosomes, extraequatorial chromosomes, early segregation, laggard chromosomes, chromosome stickiness, asynchronous meiosis and deviant cytokinesis (monad, dyads, triads), was observed, which could be the cause of 2n pollen formation. Moreover, we confirmed a relationship among the length–width ratio of flower buds, stylet length and microsporogenesis. This result suggested that we can immediately determine the microsporogenesis stages by phenotypic characteristics, which may be applicable to breeding advanced germplasm in Camellia oleifera.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01002-5.