Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp.

Journal of Experimental Botany, Jun 2011

Polyploidy is an important evolutionary phenomenon but the mechanisms by which polyploidy arises still remain underexplored. There may be an environmental component to polyploidization. This study aimed to clarify how temperature may promote diploid gamete formation considered an essential element for sexual polyploidization. First of all, a detailed cytological analysis of microsporogenesis and microgametogenesis was performed to target precisely the key developmental stages which are the most sensitive to temperature. Then, heat-induced modifications in sporad and pollen characteristics were analysed through an exposition of high temperature gradient. Rosa plants are sensitive to high temperatures with a developmental sensitivity window limited to meiosis. Moreover, the range of efficient temperatures is actually narrow. 36 °C at early meiosis led to a decrease in pollen viability, pollen ectexine defects but especially the appearance of numerous diploid pollen grains. They resulted from dyads or triads mainly formed following heat-induced spindle misorientations in telophase II. A high temperature environment has the potential to increase gamete ploidy level. The high frequencies of diplogametes obtained at some extreme temperatures support the hypothesis that polyploidization events could have occurred in adverse conditions and suggest polyploidization facilitating in a global change context.

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Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp.

Journal of Experimental Botany Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp. Yann Pe´ crix 1 Ge´ raldine Rallo 0 1 He´ le` ne Folzer 1 Mireille Cigna 1 Serge Gudin 1 Manuel Le Bris 1 0 Meilland International , Domaine Saint-Andre ́ , F-83340 Le Cannet des Maures , France 1 Institut M e ́diterrane ́ en d'Ecologie et de Pale ́ oe ́ cologie UMR CNRS 6116, Universite ́ Paul C e ́zanne Aix-Marseille III , Av. Escadrille Normandie-Niemen, F-13397 Marseille , France Polyploidy is an important evolutionary phenomenon but the mechanisms by which polyploidy arises still remain underexplored. There may be an environmental component to polyploidization. This study aimed to clarify how temperature may promote diploid gamete formation considered an essential element for sexual polyploidization. First of all, a detailed cytological analysis of microsporogenesis and microgametogenesis was performed to target precisely the key developmental stages which are the most sensitive to temperature. Then, heat-induced modifications in sporad and pollen characteristics were analysed through an exposition of high temperature gradient. Rosa plants are sensitive to high temperatures with a developmental sensitivity window limited to meiosis. Moreover, the range of efficient temperatures is actually narrow. 36 C at early meiosis led to a decrease in pollen viability, pollen ectexine defects but especially the appearance of numerous diploid pollen grains. They resulted from dyads or triads mainly formed following heat-induced spindle misorientations in telophase II. A high temperature environment has the potential to increase gamete ploidy level. The high frequencies of diplogametes obtained at some extreme temperatures support the hypothesis that polyploidization events could have occurred in adverse conditions and suggest polyploidization facilitating in a global change context. Diploid gamete; meiosis; microsporogenesis; pollen viability; polyploidization; Rosa sp; temperature Introduction In the context of global climate change, key climatic parameters expected to be adversely affected are average surface temperatures, atmospheric CO2 concentrations, UV radiation, and rainfall regimes. Concerning the temperature, over the past 100 years, global air temperatures have increased by 0.7460.18 C and an increasing rate of warming has taken place over the last 25 years. Overall, global climate change models predict a temperature increase ranging from 1.8 C to 4 C over the next century. An additional characteristic of future climatic change is, on a local scale, the increase in frequency and intensity of hot extremes and heat-wave events, even occurring with relatively small mean climate changes (Solomon et al., 2007) . Obviously, such environmental changes would have drastic effects on plant growth and development, species distribution, and diversity (Minorsky, 2002; Porter and Semenov, 2005) . On a whole plant level, elevated temperature induces multiple physiological disruptions, affecting both vegetative and reproductive processes. The sexual reproductive stage seems to be particularly vulnerable, resulting in a crop yield decrease for the most negative expected effect (Peng et al., 2004; Tubiello et al., 2007; Barnaba´ s et al., 2008; Hedhly et al., 2009; Zinn et al., 2010) . To unravel the causes of such a crop yield decrease, previous works describing the impact of elevated temperature strain focused on different sexual plant reproduction stages: (i) flower initiation and development, (ii) gametophyte development, (iii) pollination and fertilization, or (iv) embryo development and seed maturation. As far as flower initiation and development are tetraploid crops (Veilleux, 1985; Ramanna and Jacobsen, concerned, high temperature exposure reduced the number 2003) . of floral buds due to less ramified inflorescences or floral The aim of the present work was to characterize the bud abortion on many species (Guilioni et al., 1997; impact of elevated temperature on male gametophyte Bjo¨ rkman and Pearson, 1998; Warner and Erwin, 2005) . formation in Rosa focusing on meiosis, diploid gamete Heat-stressed flowers exhibited changes in development like formation, and pollen grain characteristics. In this genus, petal and stamen length decrease, anther dehiscence defects diploid gamete production is suspected to be particularly or pistil hyperplasia (Takeoka et al., 1991; Beppu et al., high and environmentally dependent because of (i) a large 2001; Porch and Jahn, 2001, Koti et al., 2005; Sato et al., range of ploidy levels between species from 2n¼2x¼14 2006). (e.g. R. wichurana Cresp.) to 2n¼8x¼56 (e.g. R. acicularis With regard to both the male and female gametophytic Lindl.) (Yokoya et al., 2000; Roberts et al., 2009) , (ii) more response to high temperature, many studies showed than half of the species are polyploids (Vamosi and a reduction in the number of produced or released pollen Dickinson, 2006) , (iii) the existence of stable odd polygrains (Koti et al., 2005; Sato et al., 2006), a decrease of ploidy species (e.g. pentaploid R. canina), and (iv) the pollen viability (Porch and Jahn, 2001) , changes in pollen great variation of pollen ploidy level in rose plants grown wall structure (Porch and Jahn, 2001; Koti et al., 2005) , and in the field under variable climatic conditions (Crespel female fertility (Peet et al., 1998; Young et al., 2004). High et al., 2006). temperature exposure could also influence pollination by inducing a decrease of pollen germination rate and pollen tube growth, and fertilization (Cross et al., 2003; Young Materials and methods et al., 2004; Koti et al., 2005). Subsequent events from embryogenesis to seed maturation were also shown to be Plant materials potentially sensitive to heat exposure resulting in progenies The experiments were carried out in wild species or rose cultivars: with a leaf area increase or delayed bud forming and six diploid rose plants (R. chinensis ‘Old blush’, R. wichurana, dehardening (Lacey and Herr, 2000; Johnsen et al., 2005a, b) . R. hybrida ‘Camelia’, R. hybrida ‘The fairy’, R. hybrida ‘HW336’, Meiosis, a key step of gamete development, might also be bRa.chcayrbar’i,daR.‘ HhyWbr7id’)a a‘nMdontharceoe’,tRet.rahpylboriiddao‘nVeisva(rRa.is’h)ywbreirdeau‘sBeldacink susceptible to environmental factors as previously shown in the analysis of pollen size and ploidy level. The detailed analysis of Capsicum annuum (Erickson and Markhart, 2002; Zhang heat treatment effects was exclusively performed on recurrent et al., 2003) . In spite of the severity of stress-induced flowering R. hybrida ‘HW336’, a diploid interspecific hybrid which meiotic anomalies, the meiotic response to abiotic stress still was obtained from a cross between a dihaploid rose, R. hybrida remains poorly documented. Meiotic anomalies are often ‘(HCr1e9s0p’e(l Meteayln.,e2t0e0t6a)l..,A1ll99p4la),ntasnwdetrheecduilptilvoaidtedspiencigersoRw.thwcichhaumrabnear chromosome mis-segregation, spindle mis-orientation or conditions: the temperature was set at 2460.5 C and a relative cytokinesis defects. Following a stress, gametes can abort humidity at 6065%. Light (PPFD c. 200 lE m 2 s 1 at canopy or exhibit unusual ploidy levels such as aneuploidy or have level) was produced by Sylvania T8 luxline plus F36W840 tubes a somatic chromosome number (i.e. diploid gametes) (Sylvania, Raunheim, Germany) for a 16 h photoperiod. Plants (Veilleux and Lauer, 1981; McHale, 1983; Negri and pweearte:cglraoyw4n0:i5n0:51.00 lbypovtoslc.)osnutapipnlienmgepnetaedt mwiixthtu2re5 (gwohfiteslopwea-tr:eblelaacske Lemmi, 1998; Zhang et al., 2003; Fuzinatto et al., 2008 ; fertilizer (Osmocote, 9-14-19). Twice a day, each plant was Dewitte et al., 2010) often resulting in a decrease in irrigated with 500 ml of water. fertilization rate. However, diploid gamete production can result in extraordinary cross-hybridization opportunities. Heat treatments Indeed, in polyploid formation by sexual polyploidization, Temperature treatments were conducted in a phytotronic chamber diploid gamete production is considered to be the dominant (Economic Premium; Snijders Scientific, Tilburg, Netherlands). route (Bretagnolle and Thompson, 1995; Ramsey and Except for temperature, the conditions in the phytotron were Schemske, 1998; Soltis et al., 2004; Soltis and Soltis, 2009) . adjusted to those of the growth chamber (60% relative humidity, Polyploidization is widely accepted as a source of species PPFD 200 lE m 2 s 1, 16 h photoperiod, and watering diversification and speciation in plants (Otto and Whitton, ccoonntdriotilo),n3s)0. TCem,3p3eraCtuorer t3r6eatCmefnotr m48odha.lBitieefsorweetrreea2t4meCnt, (aflsotrhael 2000; Rieseberg and Willis, 2007; Wood et al., 2009; Soltis buds were individually measured in planta to estimate their and Soltis, 2009; Soltis et al., 2009) . Most plants, if not all, developmental stage. Following treatment, flower buds were have a polyploid ancestor (Masterson, 1994; Adams and analysed immediately or at the anthesis stage after a period of Wendel, 2005; Cui et al., 2006) and, recently, it was growth in the growth chamber. estimated that 15% of angiosperm and 31% of fern speciation events are associated with an increase in ploidy Microscopy analysis (Wood et al., 2009). In plant breeding, interspecific Before microsporogenesis, the anthers were fixed overnight in hybridization has often been limited by cross barriers, ethanol–acetic acid fixative solution (3:1 v/v) at 4 C and stored in which are frequently due to ploidy level differences. 70% ethanol until analysis. Breeding strategies based on diploid gametes are currently (19M96e)iotpircoficegduurerse wfoerre porbespearrviendg ftohlleowwihnoglea manoddrioeficeidumRoossf eetaachl. considered as a key feature to overcome these barriers for flower bud. Meiotic chromosome spreading of the prophase and the introgression of diploid wild germplasm traits in late meiotic stages was observed after anther digestion with 1 N HCl and staining with DAPI (4’,6-diamidino-2-phenylindole) at 1 lg ml 1 in PBS at pH 7. Sporad figures and pollen grains were examined after squashing the whole androecium of each flower bud in Alexander staining solution (Alexander, 1969) . Viable pollen exhibited a purple stain rounded with green sporoderm whereas aborted pollen failed to stain. Pollen viability was determined from at least 500 pollen grains per flower. The size of c. 1000 viable pollen grains per flower was measured with an ocular micrometer. For both light microscopy and DAPI staining analysis, an Axioskop 40 microscope (Carl Zeiss, Thornwood, NY, USA) equipped with epifluorescence illumination was used. Images were taken using a DXM1200F camera driven by ACT-1 software (Nikon, Melville, NY, USA). For scanning electron microscopy analysis, fixed pollen grains were rinsed with ethanol and air-dried. Samples were sputtercoated with gold, using a Cressington 108 auto sputter coater (Cressington Scientific Instruments, Watford, England) and observed under an ESEM Philips XL 30 microscope (Philips, Eindhoven, Netherlands) at an accelerating voltage of 20 kV. Statistical analysis Statistical analysis of data was carried out using the R statistical environment (R Development Core Team, 2007). For the classification and regression tree (CART), the analysis was performed as described in the tree package (function tree) (Breiman et al., 1984) . Concerning diploid pollen grain production and pollen viability, the results presented in this paper are means 6SE. Since these data failed the Shapiro test for normalcy (shapiro.test) and Bartlett test for homogeneity of variances (bartlett.test), the non-parametric Wilcoxon rank-test (wilcox.test) was used to indicate which groups are statistically different from the others. Each result is issued from at least three independent experiments and the number of analysed flower buds in the different experiments is specified in each figure legend. Results Male sporogenesis and gametogenesis characterization in Rosa Cytological events: Firstly to investigate male sporogenesis and gametogenesis in Rosa and to discriminate the different meiotic events, DAPI-stained male meiocytes were prepared from 327 buds of diploid R. hybrida ‘HW336’ grown at 24 C under standard culture conditions (Fig. 1). At the early leptotene stage, the 14 chromosomes began to condense and appeared as threads (Fig. 1A). At the zygotene stage, chromosomes underwent synapsis (Fig. 1B) up to the pachytene stage with fully synapsed homologous chromosomes (Fig. 1C). At the diplotene stage (Fig. 1D), desynapsed chromosomes continued to condense and their shortening was clearly visible at diakinesis (Fig. 1E), with seven bivalents linked by chiasmata. At metaphase I, bivalents positioning on metaphase plate was followed by anaphase I with homologous chromosome separation (Fig. 1F, G). At metaphase II, two groups of seven chromosomes were positioned on two new perpendicularly oriented metaphasic plates (Fig. 1H). Anaphase II was characterized by individual chromatid separation leading to four polar sets of seven chromatids at telophase II (Fig. 1I). Each set of chromatids was progressively decondensed leading to a tetrad with four haploid interphase nuclei (Fig. 1J). Cytokinesis occurred, releasing unicellulate haploid microspore (Fig. 1K), followed by rounds of mitosis leading to bicellulate pollen with a vegetative nucleus and a generative cell (Fig. 1L). Since no abnormal meiosis was observed, this cultivar was definitely chosen to analyse the effect of temperature on male meiosis. Flower bud morphological and meiosis stage correlation: In addition to the previous characterization, flower morphological markers which could possibly be representative of the different meiosis stages were searched in order to allow heat stresses to be applied subsequently at accurate cytological stages. No obvious morphological variations were detected, so a correlation between hypanthium size and microsporogenesis stages was ascertained: For every 327 flower buds, hypanthium length and width were measured and associated with the cytological characterization. These data were subjected to classification and regression tree (CART) analysis (Breiman et al., 1984) . By this method, both hypanthium length and width allowed five stages to be discriminated with a high degree of significance (Fig. 2): microsporogenesis was divided into three steps: premeiosis (PM), early meiosis (eM) (from preleptotene stage to diplotene), and meiosis (M) (from diakinesis to second meiotic division) and microgametogenesis was split into two steps: tetrad (T) and both immature and mature pollen (P). Reliabilities of microsporogenesis and microgametogenesis step predictions were 98%, 95%, and 72% for the PM, eM, and M stages and 77% and 98% for the T and P stages, respectively. Pollen size and ploidy level correlation: The distribution of mean diameter of pollen grains was established by the measurement of c. 1000 pollen grains from both six different diploid cultivars or wild species and three tetraploid ones, all grown in growth chamber conditions. The diameter of viable pollen grains of diploid individuals ranged from 18 lm to 36 lm with a mean of 26.862.6 lm. Those of tetraploid individuals ranged from 24 lm to 42 lm with a mean of 35.463.2 lm (Fig. 3). The two distributions overlapped each other between 24 lm to 36 lm. As only 0.14% of haploid pollen grains issued from diploid individuals showed a diameter higher than 36 lm, pollen grains were considered as diploid from 36 lm diameter threshold in subsequent analyses. Temperature effect analysis: Subsequent analyses of high temperature effects were carried out in diploid R. hybrida ‘HW336’ which followed the general pollen grain diameter distribution issued from diploid individuals as previously described: The mean pollen grain diameter was 26.063.3lm and 0.2% of pollen grains revealed a diameter higher than 36 lm. Developmental sensitivity: To determine the stages of microsporogenesis and microgametogenesis sensitive to high temperature, flower buds at different developmental stages estimated by hypanthium size, were exposed to 36 C for 48 h (Fig. 4). Depending on the developmental stage, a high diploid pollen production was induced. Diploid pollen production was significantly higher than the control when a high temperature was applied during the pre-meiotic and meiotic stages: 7.4% and 10.3% (P <0.001) at the PM and M stages, respectively, and the highest response was at the eM stage with 24.5% (P <0.001). When the high temperature exposure was applied after meiosis during the T stage, the percentage of diploid pollen was very low and nearly null at the P stage. Concerning pollen viability, high temperature treatments applied during meiotic stages (eM, M) resulted in an increase in the number of small, collapsed (Fig. 9B) and unstained pollen grains considered as aborted (Fig. 5). Pollen abortion was significantly higher than the control in heat-treated buds during meiosis. Indeed, the rate of viable pollen grain was 43.4% and 59.2% (P <0.001) at the eM and M stages, respectively, whereas in the control, viable pollen production reached 75.6%. No significant effect of heat treatment was observed in either the pre-meiotic stage (PM) or in the post-meiotic stages (T, P). Temperature intensity effects: The effect of heat intensity on diploid pollen production and pollen abortion was investigated by performing two additional heat treatments at 30 C and 33 C for 48 h at the eM stage. Whatever the temperature, diploid pollen production was significantly higher than the control (P <0.001) with a gradual response: At 30 C, 33 C, and 36 C, diploid pollen grain productions were respectively 1.1%, 9.7%, and 24.5% versus 0.2% in the control (Fig. 6). Concerning pollen viability, the higher the temperature was, the lower the rate of viable pollen was (Fig. 7). It was significantly lower than in the control after 33 C and 36 C treatments, with 60.5% and 43.4% of viable pollen, respectively. Meiotic abnomalies To characterize the cytological events induced by heat treatment best, resulting in pollen abortion or in diploid pollen formation, microscopic investigations were performed. Buds from plants exposed to 36 C at the eM stage were dissected and the stamens were stained. At the sporad stage, tetrads but also atypical sporads like dyads, triads, and polyads were clearly discernible (Fig. 8A, D, E, F). Following heat treatment in meiocytes at telophase II, particular spindle orientations with a parallel or tripolar shape were observed (Fig. 8B) versus a roughly perpendicular shape in the control. Moreover, cytomixis events defined as chromosome exchanges between meiocytes through cytomictic channels were detected at the prophase stage (Fig. 8G). Pollen morphology In buds which were heat-treated at 36 C at the eM stage, the morphology of the pollen was observed by scanning electron microscopy. The pollen produced in the control was homogenous, tricolpate with elongated and slit-like colpi and the ectexine was weakly striated (Fig. 9A, C, E). After heat treatment, the morphology of the pollen was sometimes affected in colpi number (Fig. 9D) and variations in ectexine deposition were also recorded, resulting in less striated structure (Fig. 9F). Pollen clustering, revealed by unreleased pollen grains remaining associated with tetrads, triads or dyads, was also observed (Fig. 9B). Discussion This study has demonstrated the effect of a range of high temperatures on male gametophyte development in Rosa. Meiosis, pollen viability, and pollen wall structure have proved to be affected by heat exposure. High temperature and pollen viability decrease In the present study, high temperature exposure affected pollen viability both at a dosage- and developmental stage-dependent way. Heat-treated flower buds produced fewer viable pollen grains and the higher the temperature the lower the viability rate. Such heat susceptibility of microsporogenesis leading to pollen abortion was already suggested in various species like Phaseolus vulgaris (Porch and Jahn, 2001) , Capsicum annuum (Erickson and Markhart, 2002) , Oryza sativa (Cao et al., 2008) or Solanum lycopersicum (Iwahori, 1965; Sato et al., 2002) . However, in Rosa, this response concerned a narrow window of development which was limited to microsporogenesis and, specifically, to early meiosis. Indeed, only the buds which were heat-treated during prophase showed a decrease in pollen viability. This observed heatinduced pollen abortion could have both a sporophytic and a gametophytic origin. Indeed, as all the anther wall tissues, such as the differentiated stomium, tapetum, middle layers or endothecium, are present at the moment of meiosis (Goldberg et al., 1993) , heat stress applied at this stage could alter the function of these sporophytic tissues. Tapetum, located at the interface between gametophytic and sporophytic tissues, is known to supply components that are essential for male gametophyte development: for instance, callase or sporopollenin precursors are necessary for callose dissolution and exine formation, respectively (Scott et al., 2004). Lack of callase or a temporal shift in its activity can affect the formation of the normal microspore cell wall and in fine the fertility level: in Glycine max, Jin et al. (1997) reported that a lack of callase production could lead to complete male sterility. In Nicotiana tabacum, Worrall et al. (1992) demonstrated that early callose degradation was sufficient to cause male sterility. Generally, anther wall tissue degeneration following heat treatments has been related to an increase in pollen sterility and an alteration in pollen wall structure (Porch and Jahn, 2001; Suzuki et al., 2001; Sato et al., 2002; Koti et al., 2005) . The trophic role of sporophytic tissues in gametophyte development could also be imputed, as reported by Pressman et al. (2002) in Solanum lycopersicum: high temperature exposure reduced starch concentration in the anther, resulting in a decrease in soluble sugars in both the anther and the mature pollen. Some heat-induced defects reported in this study, including pollen clustering, pollen abortion or abnormal ectexine ornamentation, effectively suggest a sporophyte dysfunctioning. In addition to a sporophytic origin, the decrease in pollen viability in Rosa can also be attributed to a direct gametophyte response, revealed by aberrant sporads in heat-treated buds. The cytomixis events observed in prophase could partially explain such sporads. Indeed, in plants, cytoplasm, organelles, and, in some cases, chromatin exchanges could occur between meiocytes through cytomictic channels derived from plasmodesmata. Occasionally, during callose deposition, some plasmodesmata are not obstructed, increase in size, and form channels connecting meiocytes. These channels allow symplasmic domain forming and are believed to promote synchrony within the microsporocyte mass (Heslop-Harrison, 1966) . Cytomixis events involving chromosome exchanges in meiosis (Malallah and Attia, 2003; Lattoo et al., 2006; Sheidai, 2008; Singhal and Kumar, 2008a, b; Mursalimov et al., 2010) were considered as a cause of abnormal sporads like unbalanced tetrads or polyads which are responsible for pollen sterility (Soodan and Wafai, 1987) . The occurrence of cytomixis is both under genetic control (Bellucci et al., 2003) and susceptible to environmental factors such as high diurnal temperature (Narain, 1979; Basavaiah and Murthy, 1987) . However, the aberrant sporads leading to a decrease in pollen viability in Rosa could be attributed not only to cytomixis events but also to other irregularities occurring during meiosis such as chromosomal cohesion, irregular pairing, and segregation (Sapre and Deshpande, 1987; Bellucci et al., 2003) . High temperature and diploid pollen production Another original response to high temperatures in Rosa is a high production of diploid pollen. Diploid pollen production was detected by pollen size measurement and by cytological observation at the sporad stage. Because of the close correlation between pollen DNA content and cell volume, the occurrence of giant pollen grains is often used as an indicator of diploid pollen production (Altmann et al., 1994) . Such a relationship, firstly established in Rosa by Jacob and Pierret (2000) , was confirmed here by comparing pollen size distribution between diploid and tetraploid cultivars. According to species, diploid pollen can be formed by pre- or post-meiotic chromosome doubling but are mainly originated from meiotic dysfunctioning such as a defect in chromosome synapsis, omission of the first or second meiotic division, defect in spindle positioning or abnormal cytokinesis (Bretagnolle and Thompson, 1995) . In this study, in Rosa, diploid pollen production is solely due to irregularities occurring during meiosis. Two potential meiotic defects were identified: firstly, cytomixis events, as described above, can create variation in the chromosome number of the microspores and have been described as an effective mechanism for diploid gamete formation (Falistocco et al., 1995; Ghaffari, 2006) . Secondly, the most frequent meiotic irregularity observed in this study, is the mis-orientation of meiotic spindle in meiosis II. Therefore, this type of abnormality which results in dyad (fused or parallel spindle) or triad (tripolar spindle) formation is considered as the main cause of diploid pollen formation in Rosa. Fused, parallel, or tripolar spindles at meiosis II have already been described in other species such as Solanum sp. (Watanabe and Peloquin, 1993; Peloquin et al., 1999) , Ipomoea batatas (Becerra Lopez-Lavalle and Orjeda, 2002) , Populus tomentosa (Zhang and Kang, 2010) , Lotus tenuis (Negri and Lemmi, 1998) or Medicago sativa (Tavoletti et al., 1991) . These spindle mis-orientations were systematically associated with diploid pollen formation as well. Moreover, the present study ascertains the close relationship between diploid pollen formation and environment. Numerous exogenous factors were thought to give rise to diploid pollen. In general, they were issued from a multifactorial combination involving biotic factors such as herbivory, wounding, and disease or abiotic factors such as altitude, latitude, water and nutrient stress (reviewed in Bretagnolle and Thompson, 1995; Ramsey and Schemske 1998) . In Rosa, diploid pollen production had been previously detected in different field-grown cultivars regularly analysed during a three-year period (Crespel et al., 2006) . The frequencies of diploid pollen were highly variable seasonally, inter-annually, and between genotypes. These variations were attributed to a combination of environmental and genetic factors. In other species, some reports pointed out the relevance of temperature as the environmental factor inducing diploid pollen production. Nevertheless, the temperature effects on diploid pollen production resulted from experiments that were carried out on long-term cultures or with a large range of temperatures and few studies were performed under controlled culture conditions (Negri and Lemmi, 1998; Zhang et al., 2003). Temperature as a single and efficient environmental factor inducing a high frequency of diploid pollen production has been highlighted here. The plants were sensitive to high temperatures and the range of efficient temperatures is actually narrow. Moreover, the sensitivity window is limited to meiosis. The coincidence of accurate developmental flower stage and precise high temperatures are necessary to induce diploid pollen production. In the field or under uncontrolled environmental conditions, such a relationship is quite difficult to recognize and could account for the large fluctuations observed in fieldgrown roses (Crespel et al., 2006) . In plant evolution, because of its key role in sexual polyploidization or whole genome duplication events, diploid gamete formation is considered as an important component in diversification and speciation. In plant domestication, introgressing wild species traits into cultivars to extend genetic diversity has been investigated. However, like Rosa, many important crops such as Triticum aestivum, Gossypium hirsutum, Solanum tuberosum or Saccharum officinarum are polyploid. Interspecific hybridizations between mostly diploid wild species and polyploid crops are commonly hampered by triploid sterile or poorly fertile progeny. The control of diploid gamete production by environmental factors represents a method of choice to overcome these ploidy barriers (Veilleux, 1985; Ramanna and Jacobsen, 2003) and could be transferable to other crops. As shown in the present study, the formation of diploid gametes depends on development–environment interactions but is also known to be under strong genetic control (Ramsey and Schemske, 1998; Otto and Whitton, 2000). For only a couple of years, some genes involved in the production of diploid pollen have been identified, mostly by Arabidopsis thaliana mutant screening. For instance, the Atps1 mutant shows parallel spindle orientation solely in male meiosis II (d’Erfurth et al., 2008) . Osd1 and tam-2 mutants are characterized by the omission of male meiosis II and both male and female diploid gametes were observed, leading to the duplication of the ploidy level in the progeny (d’Erfurth et al., 2009; Wang et al., 2010) . The function of these genes and the regulation of their expression still remain poorly known. Rosa, with its heat-inducing diploid pollen formation, might be an excellent tool to target new genes or regulation pathways involved in diploid pollen formation by differential expression analyses. Such information might provide new tools for plant breeding and enlarge our knowledge about sexual polyploidization mechanisms. Acknowledgements YP received a PhD grant co-funded by the French PACA region and Meilland International (Le Cannet des Maures, France). We thank M Bendahmane and O Raymond (ENS Lyon, France) for their critical reading of the manuscript, R Mercier (INRA Versailles, France) for his helpful comments in meiosis analysis, R Verlaque, R Notonier, and A Tonetto (Service commun de microscopie Universite´ de Provence, France) for their technical assistance in SEM and F Torre (IMEP Marseille, France) in statistical analysis. We are also grateful to L Crespel and Meilland International for providing plant material. affected by the temperature during zygotic embryogenesis and seed maturation. Plant, Cell and Environment 28, 1090–1102. Koti S, Reddy KR, Reddy VR, Kakani VG, Zhao DL. 2005. Interactive effects of carbon dioxide, temperature, and ultraviolet-B radiation on soybean (Glycine max L.) flower and pollen morphology, pollen production, germination, and tube lengths. Journal of Experimental Botany 56, 725–736. Lacey EP, Herr D. 2000. Parental effects in Plantago lanceolata L. III. Measuring parental temperature effects in the field. Evolution 54, 1207–1217. Lattoo SK, Khan S, Bamotra S, Dhar AK. 2006. Cytomixis impairs meiosis and influences reproductive success in Chlorophytum comosum (Thunb) Jacq.: an additional strategy and possible implications. Journal of Biosciences 31, 629–637. Malallah GA, Attia TA. 2003. Cytomixis and its possible evolutionary role in a Kuwaiti population of Diplotaxis harra (Brassicaceae). Botanical Journal of the Linnean Society 143, 169–175. Masterson J. 1994. Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science 264, 421–424. McHale NA. 1983. Environmental induction of high frequency 2 n pollen formation in diploid. Solanum. Canadian Journal of Genetics and Cytology 25, 609–615. Meynet J, Barrade R, Duclos A, Siadous R. 1994. Dihaploid plants of roses (Rosa3 hybrida, cv. Sonia) obtained by parthenogenesis Induced using irradiated pollen and in vitro culture of immature seeds. Agronomie 14, 169–175. Minorsky PV. 2002. Global warming: effects on plants. Plant Physiology 129, 1421–1422. Mursalimov SR, Baiborodin SI, Sidorchuk YV, Shumny VK, Deineko EV. 2010. Characteristics of the cytomictic channel formation in Nicotiana tabacum L. pollen mother cells. Cytology and Genetics 44, 14–18. Narain P. 1979. Cytomixis in the pollen mother cells of Hemerocallis Linn. Current Science 48, 996–998. Negri V, Lemmi G. 1998. Effect of selection and temperature stress on the production of 2 n gametes in. Lotus tenuis. Plant Breeding 117, 345–349. Pressman E, Peet MM, Pharr DM. 2002. The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers. Annals of Botany 90, 631–636. R Development Core Team. 2007. R: a language and environment for statistical computing. Vienna: Austria R Foundation for Statistical Computing. Ramanna MS, Jacobsen E. 2003. Relevance of sexual polyploidization for crop improvement: a review. Euphytica 133, 3–18. Ramsey J, Schemske DW. 1998. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annual Review of Ecology and Systematics 29, 467–501. Rieseberg LH, Willis JH. 2007. Plant speciation. Science 317, 910–914. Roberts AV, Gladis T, Brumme H. 2009. DNA amounts of roses (Rosa L.) and their use in attributing ploidy levels. Plant Cell Reports 28, 61–71. Ross KJ, Fransz P, Jones GH. 1996. A light microscopic atlas of meiosis in. Arabidopsis thaliana. Chromosome Research 4, 507–516. Sapre AB, Deshpande DS. 1987. A change in chromosome number due to cytomixis in an interspecific hybrid of Coix L. Cytologia 52, 167–174. Sato S, Kamiyama M, Iwata T, Makita N, Furukawa H, Ikeda H. 2006. Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development. Annals of Botany 97, 731–738. Sato S, Peet MM, Thomas JF. 2002. Determining critical pre- and post-anthesis periods and physiological processes in Lycopersicon esculentum Mill. exposed to moderately elevated temperatures. Journal of Experimental Botany 53, 1187–1195. Scott RJ, Spielman M, Dickinson HG. 2004. Stamen structure and function. The Plant Cell 16, 46–60. Sheidai M. 2008. Cytogenetic distinctiveness of sixty-six tetraploid cotton (Gossypium hirsutum L.) cultivars based on meiotic data. Acta Botanica Croatica 67, 209–220. Singhal VK, Kumar P. 2008a. Cytomixis during microsporogenesis in Otto SP, Whitton J. 2000. Polyploid incidence and evolution. Annual the diploid and tetraploid cytotypes of Withania somnifera (L.) Dunal, Review of Genetics 34, 401–437. 1852 (Solanaceae). Comparative Cytogenetics 2, 85–92. Young LW, Wilen RW, Bonham-Smith PC. 2004. High temperature stress of Brassica napus during flowering reduces micro- and Vamosi JC, Dickinson TA. 2006. Polyploidy and diversification: megagametophyte fertility, induces fruit abortion, and disrupts seed a phylogenetic investigation in Rosaceae. International Journal of Plant production. Journal of Experimental Botany 55, 485–495. Sciences 167, 349–358. Zhang XZ, Liu GJ, Yan LY, Zhao YB, Chang RF, Wu LP. 2003. Veilleux R. 1985. Diploid and polyploid gametes in crop plants: Creating triploid germplasm via induced 2n pollen in Capsicum mechanisms of formation and utilization in plant breeding. Plant annuum L. Journal of Horticultural Science and Biotechnology 78, Breeding Reviews 3, 252–288. 84–88. Adams KL , Wendel JF . 2005 . Polyploidy and genome evolution in plants . Current Opinion in Plant Biology 8 , 135 - 141 . Alexander MP . 1969 . Differential staining of aborted and nonaborted pollen . Stain Technology 44 , 117 - 122 . Altmann T , Damm B , Frommer WB , Martin T , Morris PC , Schweizer D , Willmitzer L , Schmidt R. 1994 . Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement . Plant Cell Reports 13 , 652 - 656 . Barnaba´ s B , Ja¨ ger K , Fehe´ r A. 2008 . The effect of drought and heat stress on reproductive processes in cereals . Plant, Cell and Environment 31 , 11 - 38 . Basavaiah D , Murthy TCS . 1987 . Cytomixis in pollen mother cells of Urochloa panicoides P. Beauv. (Poaceae) . Cytologia 52 , 69 - 74 . Becerra Lopez-Lavalle LA , Orjeda G. 2002 . Occurrence and cytological mechanism of 2n pollen formation in a tetraploid accession of Ipomoea batatas (sweet potato) . Journal of Heredity 93 , 185 - 192 . Bellucci M , Roscini C , Mariani A. 2003 . Cytomixis in pollen mother cells of Medicago sativa L . Journal of Heredity 94 , 512 - 516 . Beppu K , Ikeda T , Kataoka I. 2001 . Effect of high temperature exposure time during flower bud formation on the occurrence of double pistils in 'Satohnishiki' sweet cherry . Scientia Horticulturae 87 , 77 - 84 . Bj o¨rkman T , Pearson KJ . 1998 . High temperature arrest of inflorescence development in broccoli (Brassica oleracea var . italica L.). Journal of Experimental Botany 49 , 101 - 106 . Breiman L , Friedman JH , Olshen RA , Stone CJ . 1984 . Bretagnolle F , Thompson JD . 1995 . Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants . New Phytologist 129 , 1 - 22 . Cao YY , Duan H , Yang LN , Wang ZQ , Zhou SC , Yang JC. 2008 . Effect of heat stress during meiosis on grain yield of rice cultivars differing in heat tolerance and its physiological mechanism . Acta Agronomica Sinica 34 , 2134 - 2142 . 2003. Heat-stress effects on reproduction and seed set in Linum usitatissimum L. (flax) . Plant, Cell and Environment 26 , 1013 - 1020 . Cui L , Wall PK , Leebens-Mack JH , et al. 2006 . Widespread genome duplications throughout the history of flowering plants . Genome Research 16 , 738 - 749 . d'Erfurth I , Jolivet S , Froger N , Catrice O , Novatchkova M , Mercier R. 2009 . Turning meiosis into mitosis . PLoS Biology 7 , e1000124 . d'Erfurth I , Jolivet S , Froger N , Catrice O , Novatchkova M , Simon M , Jenczewski E , Mercier R. 2008 . Mutations in AtPS1 (Arabidopsis thaliana parallel spindle 1) lead to the production of diploid pollen grains . PLoS Genetics 4 , e1000274 . 2010. Induction of 2n pollen formation in Begonia by trifluralin and N2O treatments . Euphytica 171 , 283 - 293 . Erickson AN , Markhart AH . 2002 . Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature . Plant, Cell and Environment 25 , 123 - 130 . Falistocco E , Tosti N , Falcinelli M. 1995 . Cytomixis in pollen mother cells of diploid Dactylis, one of the origins of 2 n gametes . Journal of Heredity 86 , 448 - 453 . Fuzinatto VA , Pagliarini MS , Valle CB . 2008 . Evaluation of microsporogenesis in an interspecific Brachiaria hybrid (Poaceae) collected in distinct years . Genetics and Molecular Research 7 , 424 - 432 . Ghaffari SM . 2006 . Occurrence of diploid and polyploid microspores in Sorghum bicolor (Poaceae) is the result of cytomixis . African Journal of Biotechnology 5 , 1450 - 1453 . Goldberg RB , Beals TP , Sanders PM . 1993 . Anther development: basic principles and practical applications . The Plant Cell 5 , 1217 - 1229 . Guilioni L , Wery J , Tardieu F. 1997 . Heat stress-induced abortion of buds and flowers in pea: is sensitivity linked to organ age or to relations between reproductive organs? Annals of Botany 80 , 159 - 168 . Hedhly A , Hormaza JI , Herrero M. 2009 . Global warming and sexual plant reproduction . Trends in Plant Science 14 , 30 - 36 . Heslop-Harrison J. 1966 . Cytoplasmic connexions between angiosperm meiocytes . Annals of Botany 30 , 221 - 222 . Iwahori S. 1965 . High temperature injuries in tomato . IV. Development of normal flower buds and morphological abnormalities of flower buds treated with high temperature . Journal of the Japanese Society for Horticultural Science 34 , 33 - 41 . Jacob Y , Pierret V. 2000 . Pollen size and ploidy level in the genus . Rosa. Acta Horticulturae 508 , 289 - 292 . Jin W , Horner HT , Palmer RG . 1997 . Genetics and cytology of a new genic male-sterile soybean [ Glycine max (L.) Merr.]. Sexual Plant Reproduction 10 , 13 - 21 . Johnsen Ø Daehlen OG , Østreng G , Skrøppa T. 2005 . a. Daylength and temperature during seed production interactively affect adaptive performance of Picea abies progenies . New Phytologist 168 , 589 - 596 . Crespel L , Ricci SC , Gudin S. 2006 . The production of 2 n pollen in rose . Euphytica 151 , 155 - 164 . Johnsen Ø Fossdal CG , Nagy N , Mølmann J , Daehlen OG , Skrøppa T. 2005b . Climatic adaptation in Picea abies progenies is Peet MM , Sato S , Gardner RG . 1998 . Comparing heat stress effects on male-fertile and male-sterile tomatoes . Plant, Cell and Environment 21 , 225 - 231 . Peloquin SJ , Boiteux LS , Carputo D. 1999 . Meiotic mutants in potato . Valuable variants. Genetics 153 , 1493 - 1499 . Peng SB , Huang JL , Sheehy JE , Laza RC , Visperas RM , Zhong XH , Centeno GS , Khush GS , Cassman KG . 2004 . Rice yields decline with higher night temperature from global warming . Proceedings of the National Academy of Sciences, USA 101 , 9971 - 9975 . Porch TG , Jahn M. 2001 . Effects of high-temperature stress on microsporogenesis in heat-sensitive and heat-tolerant genotypes of Phaseolus vulgaris . Plant, Cell and Environment 24 , 723 - 731 . Singhal VK , Kumar P. 2008b. Impact of cytomixis on meiosis, pollen viability and pollen size in wild populations of Himalayan poppy (Meconopsis aculeata Royle) . Journal of Biosciences 33 , 371 - 380 . Solomon S , Qin D , Manning M , Chen Z , Marquis M , Averyt KB , Tignor M , Miller HL . 2007 . Climate change 2007: the physical science basis. Contribution of working group 1 to the fourth assessment report of the intergovernmental panel on climate change. Soltis DE , Albert VA , Leebens-Mack J , Bell CD , Paterson AH , Zheng CF , Sankoff D. 2009 . dePamphilis CW , Wall PK , Soltis PS . 2009. Polyploidy and Angiosperm diversification . American Journal of Botany 96 , 336 - 348 . Porter JR , Semenov MA . 2005 . Crop responses to climatic variation . Soltis DE, Soltis PS , Tate JA. 2004 . Advances in the study of Philosophical Transactions of the Royal Society B 360 , 2021 - 2035 . polyploidy since plant speciation . New Phytologist 161 , 173 - 191 . Warner RM , Erwin JE . 2005 . Naturally occurring variation in high temperature induced floral bud abortion across Arabidopsis thaliana accessions . Plant, Cell and Environment 28 , 1255 - 1266 . Watanabe K , Peloquin SJ . 1993 . Cytological basis of 2 n pollen formation in a wide range of 2 x, 4 x, and 6 x taxa from tuber-bearing Solanum species . Genome 36 , 8 - 13 . Wood TE , Takebayashi N , Barker MS , Mayrose I , Greenspoon PB , Rieseberg LH . 2009 . The frequency of polyploid speciation in vascular plants . Proceedings of the National Academy of Sciences, USA 106 , 13875 - 13879 . Worrall D , Hird DL , Hodge R , Paul W , Draper J , Scott R. 1992 . Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco . The Plant Cell 4 , 759 - 771 . Yokoya K , Roberts AV , Mottley J , Lewis R , Brandham PE . 2000 . Nuclear DNA amounts in roses . Annals of Botany 85 , 557 - 561 . Soltis PS , Soltis DE. 2009 . The role of hybridization in plant speciation . Annual Review of Plant Biology 60 , 561 - 588 . Soodan AS , Wafai BA . 1987 . Spontaneous occurrence of cytomixis during microsporogenesis in almond (Prunus amygdalus Batsch) and peach (P. persica Batsch) . Cytologia 52 , 361 - 364 . Suzuki K , Takeda H , Tsukaguchi T , Egawa Y. 2001 . Ultrastructural study on degeneration of tapetum in anther of snap bean (Phaseolus vulgaris L.) under heat stress . Sexual Plant Reproduction 13 , 293 - 299 . Takeoka Y , Hiroi K , Kitano H , Wada T. 1991 . Pistil hyperplasia in rice spikelets as affected by heat stress . Sexual Plant Reproduction 4 , 39 - 43 . Tavoletti S , Mariani A , Veronesi F. 1991 . Cytological analysis of macro- and microsporogenesis of a diploid alfalfa clone producing male and female 2 n gametes . Crop Science 31 , 1258 - 1263 . Tubiello FN , Soussana JF , Howden SM . 2007 . Crop and pasture response to climate change . Proceedings of the National Academy of Sciences, USA 104 , 19686 - 19690 . Veilleux RE , Lauer FI . 1981 . Variation for 2n pollen production in clones of Solanum phureja Juz and Buk . Theoretical and Applied Genetics 59 , 95 - 100 . Zhang Z , Kang X. 2010 . Cytological characteristics of numerically unreduced pollen production in Populus tomentosa Carr . Euphytica 173 , 151 - 159 . Wang Y , Jha AK , Chen R , Doonan JH , Yang M. 2010 . Polyploidyassociated genomic instability in Arabidopsis thaliana . Genesis 48 , 254 - 263 . Zinn KE , Tunc-Ozdemir M , Harper JF . 2010 . Temperature stress and plant sexual reproduction: uncovering the weakest links . Journal of Experimental Botany 61 , 1959 - 1968 .

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Yann Pécrix, Géraldine Rallo, Hélène Folzer, Mireille Cigna, Serge Gudin, Manuel Le Bris. Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp., Journal of Experimental Botany, 2011, 3587-3597, DOI: 10.1093/jxb/err052