Journal of Experimental Botany, Vol. 55, No. 396, pp. 485-495, February 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production
Received 12 May 2003; Accepted 14 October 2003
1 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada
2 Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada
* To whom correspondence should be addressed. Fax: +1 306 966 4461. E-mail: bonhamp{at}duke.usask.ca
Abbreviations: HTS, high temperature stress; HSP, heat shock protein; 1WHTS, 1 week high temperature stress; 2WHTS, 2 week high temperature stress; RT-PCR, reverse transcriptase polymerase chain reaction.
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Abstract |
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High temperature stress (HTS), during flowering, decreases seed production in many plants. To determine the effect of a moderate HTS on flowering, fruit and seed set in Brassica napus, plants were exposed to a HTS (8/16 h dark/light, 18 °C night, ramped at 2 °C h–1, over 6 h, to 35 °C for 4 h, ramped at 2 °C h–1 back to 23 °C for 6 h) for 1 or 2 weeks after the initiation of flowering. Although flowering on the HTS-treated plants, during both the 1 week and 2 week HTS treatments, was equal to that of control-grown plants, fruit and seed development, as well as seed weight, were significantly reduced. Under HTS, flowers either developed into seedless, parthenocarpic fruit or aborted on the stem. At the cessation of the HTS, plants compensated for the lack of fruit and seed production by increasing the number of lateral inflorescences produced. During the HTS, pollen viability and germinability were slightly reduced. In vitro pollen tube growth at 35 °C, from both control pollen and pollen developed under a HTS, appeared abnormal, however, in vivo tube growth to the micropyle appeared normal. Reciprocal pollination of HTS or control pistils with HTS or control pollen indicated that the combined effects of HTS on both micro- and megagametophytes was required to knock out fruit and seed development. Expression profiles for a subset of HEAT SHOCK PROTEINs (HSP101, HSP70, HSP17.6) showed that both micro- and megagametophytes were thermosensitive despite HTS-induced expression from these genes.
Key words: Heat Shock Protein (HSP), high temperature stress, megagametophyte, microgametophyte, partheno carpic, pollen.
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Introduction |
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The adverse effects of high temperature stress (HTS) on plant reproduction have implications on world wide crop production systems as well as contributing to the geographical distribution of natural plant species. The sensitivity of reproductive development to HTS is not well understood. However, with a predicted temperature increase of +1.4 °C to +5.8 °C between the years 1990 and 2010 (IPCC Working Group I, 2001), a good understanding of the phenomenon will be required if the impact of these higher temperatures is to be limited and the current crop germplasm improved.
HTS during flowering results in reduced seed yield in both monocotyledon and dicotyledon plants, for example, Brassica napus (L.) (Nuttal et al., 1992; Morrison, 1993; Angadi et al., 2000), Linum usitatissimum (L.) (Gusta et al., 1997), Lycopersicon esculentum (Mill.) (Peet et al., 1998; Sato et al., 2002), Phaseolus vulgaris (L.) (Shonnard and Gepts, 1994), Triticum aestivum (L.) (Saini et al., 1983), and Zea mays (L.) (Herrero and Johnson, 1980; Carlson, 1990). The range of species adversely affected by HTS during the reproductive stage suggests that some common mechanisms may be involved in HTS-induced reduction of seed production.
B. napus plants grown throughout their life cycle at 27/17 °C light/dark were found to be almost totally sterile (Morrison, 1993). In growth chamber experiments, the greatest reduction in rate of seed production has been shown to occur when B. napus plants were exposed to a 7 d HTS treatment (35/15 °C light/dark) during early flowering (Angadi et al., 2000). These studies suggest that reductions in seed set, in HTS treated B. napus, were due to reduced gametophyte fertility or function. However, the stage of flowering most sensitive to HTS, or the gametophyte most affected by HTS have not been identified. In P. vulgaris (48 h at 35 °C or 41/21 °C light/dark; Weaver et al., 1985), L. esculentum (32/26 °C light/dark for various durations; Peet et al., 1998; Sato et al., 2002), T. aestivum (30/20 °C for 72 h; Saini and Aspinall, 1982), and Z. mays (38/32 °C for 24 or 48 h; Herrero and Johnson, 1980), pollen viability was reduced as a result of HTS, whereas in T. aestivum (Saini et al., 1983) and B. napus (28/23 °C; Polowick and Sawhney, 1987, 1988), abnormal megagametophyte development, due to HTS, has been observed. HTS affecting both gametophytes has only been reported in wheat and tomato (Saini et al., 1983; Sato et al., 2002).
Improved thermotolerance in plants has been observed with the synthesis of isoprene (Singsaas et al., 1997) or glycinebetaine (Sakamoto and Murata, 2001), the production of antioxidant enzymes such as dehydroascorbate reductase (Kubo et al., 1999) and reductions in 
-linolenic acid concentrations (Murakami et al., 2000). However, not all plants are able to use these mechanisms to improve thermotolerance. One universal response to HTS is the production of Heat Shock Proteins (HSPs, Nagao et al., 1986). Small HSPs (Malik et al., 1999; Park and Bong, 2002), HSP90 (Ludwig Muller et al., 2000) and HSP101 (Hong et al., 2000, 2001; Queitsch et al., 2000) are required for improved thermotolerance in plants. In Glycine max (L.) Merrill leaves (Kimpel and Key, 1985) and Malus domesticus Borkh. fruit (Ferguson et al., 1998), HSPs were synthesized under field conditions in response to HTS. However, HSP production in the gametophytes is not well documented and is, in some cases, contradictory (Herpen et al., 1989; Dupuis and Dumas, 1990; Winter and Sinibaldi, 1991; Mascarenhas and Crone, 1996). It is not known whether HTS induces HSP expression in B. napus gametophytes.
Although temperature stress is known to result in reduced seed production in B. napus, whether this is a result of decreased flowering, fruit abortion or disruptions in fertilization or post-fertilization events, is not known. In this paper, the effects of a moderate HTS on flower, fruit, and seed development in B. napus are reported. A population of B. napus plants were exposed to a moderate heat stress over a period of one or two weeks and the cumulative numbers of flowers, fruit, seed, parthenocarpic fruit, as well as pollen viability, pollen germinability and transcription of a subset of HSP genes in pollen and pistils were determined. As well as contributing to a better understanding of HTS effects during flowering, these data give some insights into the stages of flower–fruit–seed development that should be targeted for the development of HTS tolerant B. napus germplasm.
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Materials and methods |
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Plant growth and HTS treatment
The double haploid B. napus line, DH12075 (Gerhard Rakow, Agriculture and AgriFood Canada, Saskatoon Research Centre) was used for all experiments. DH12075 is derived from an F1 cross between the French variety Cresor and the Canadian variety Westar.
Both replicates of the experiment were carried out in GR48 growth cabinets (Conviron, Winnipeg, MB, Canada), in the Phytotron Facility (College of Agriculture, University of Saskatchewan). Two seeds were sown in each of 25–30 4.0 l pots filled with Rediearth (WR Grace and Co. Canada Ltd., Ajax, Ontario). At the two-leaf stage, the pots were thinned to one seedling per pot. Control growth conditions were 16/8 h, 23/18 °C, day/night cycles and 230–300 µE m–2 s–1 (from a combination of fluorescent and incandescent lights) at canopy level, depending upon height of the plants. When approximately 50% of the plants were flowering, the plants were randomly divided into one week high temperature stress (1WHTS), two week high temperature stress (2WHTS) and control groups. The HTS regime was as follows: daytime temperatures ramped at 2 °C h–1, from 23–35 °C over 6 h, maintained at 35 °C for 4 h then ramped back down to 23 °C for the remaining 6 h of daylight. Night temperatures remained constant at 18 °C for 8 h. After 1 or 2 weeks of HTS, the plants were returned to standard growing conditions until desiccation.
Throughout each experiment, plants were randomized weekly and were kept well watered to minimize any effects associated with drought stress. Each plant was fertilized once prior to flowering with 50 ml of 2.5 g l–1 of 20:20:20 (by vol.) fertilizer, which was applied to the soil.
The pedicel of the highest open flower on each inflorescence was tagged daily with a piece of dated tape for the duration of the experiment. Tagging in this manner allowed for the daily identification and cataloguing of all flowers exposed to HTS. The tag remained on the flower through fruit and seed development and, as such, was a means to correlate development of siliques, or lack of, with treatment. Siliques containing seeds were collected and enumerated according to the day the flower opened. Seedless, parthenocarpic siliques were not included in these data. Seed number and weight d–1 were also determined from ten randomly selected plants. Two repetitions of the experiment were carried out. The results from both repetitions showed similar patterns and therefore the data sets have been combined herein (n=51).
Pollen viability and nuclei staining
The pollen was stained using fluorescein diacetate (FDA) and the epifluorescence observed using a Zeiss Axioplan (Carl Zeiss, Thornwood, NY, USA) microscope (Heslop-Harrison and Heslop-Harrison, 1970). Starting with the first day of the heat stress, flowers of different ages were collected from the control and the HTS-treated DH12075 plants and the viability of pollen from all the anthers was determined. Flowers opening on the day of collection or one or two days prior to collection were designated as 0, 1 and 2-d-old flowers, respectively. Mature flower buds destined to open the day following collection were also collected and designated as –1-d-old flowers. Pollen was collected during the middle of the light cycle, that is after 6 h ramping, at 2 °C h–1, from 23–35 °C and 2 h at 35 °C. On each collection day, single flowers from three different plants for each age group were collected.
One or more fully-opened, 1-d-old flower(s) from each of three 8 h HTS-treated or 4 d HTS-treated or control plants were collected and the pollen subsequently stained using 2,4-diamidino-2-phenylindole (DAPI; Pechen and Keller, 1988). Nuclei were visualized using a Zeiss Axioplan microscope with a DAPI epifluorescence filterset (Pechen and Keller, 1988). Data were pooled to give the number of nuclei observed for each treatment group.
In vitro pollen tube growth
Pollen from 4–6 control-grown or HTS-treated plants (i.e. after 6 h ramping, at 2 °C h–1, from 23–35 °C and 2 h at 35 °C) was collected from flowers of approximately the same age and germinated using Hodgkins and Lyons media containing 9% sucrose and 13% polyethylene glycol (MW 4000) (Rao et al., 1992). The pollen was incubated in light and high humidity for 4 h at either 23 °C or 35 °C. Germinating pollen (those with pollen tubes greater than twice the length of the pollen grain) were counted and photographed using a Synsys digital camera (Photometrics, Huntington Beach, CA, USA) attached to a Zeiss Axioplan microscope. The digital images were processed using the MetaVue programme by Universal Imaging Group (Downingtown, PA, USA).
Reciprocal crosses between the HTS and control pollen and pistils
At least 14 immature flowers (those due to open the next day) from both the control and HTS-treated plants were emasculated by hand. Manual pollination was performed the day after emasculation using pollen from anthers of either the control or HTS-treated plants, 8 h into the light cycle on the fourth day of exposure to HTS. After pollination, the plants remained in either control or HTS conditions until the end of the HTS treatment, when all plants were grown under control conditions. Pistils were collected approximately 24 h after pollination and fixed in 1:3 glacial acetic acid:70% ethanol prior to preparation for scanning electron microscopy (SEM) as described by Hill and Lord (1987). Pollen germination and pollen tube growth in the ovary were observed with a Phillips 505 SEM (Hill and Lord, 1987).
Seven or eight pistils from each of the reciprocal crosses were left to develop for 10 d following pollination (final 3 d of HTS followed by 1 week of control temperatures) before being fixed and stored under ethanol. The total number of pistils, parthenocarpic and seed containing siliques, and the mean number of seeds per silique were recorded. Two repetitions of the experiment were carried out.
Detection of HSP mRNAs
Reverse Transcriptase PCR (RT-PCR) was used to determine the presence of HSP mRNAs in the pollen, unpollinated pistils and leaf tissue. Mature pollen was collected from control plants by agitating anthers, excised from freshly opened flowers, in 700 µl of 10% sucrose. Pollen was sedimented by centrifugation at 100 g for 2 min and the excess sucrose drawn off by pipette. The pollen pellet was resuspended in 100 µl of 10% sucrose and the aliquots incubated at 23 °C for 60 min or at 35 °C for 30 or 60 min. Immediately after incubation, the pollen was ground to a powder under liquid nitrogen. Total RNA was extracted using a Plant RNeasy kit (Qiagen, Mississauga, ON). The pollen was collected from freshly opened flowers on HTS-treated plants and the total RNA extracted immediately. Leaf discs and the unpollinated pistils of unopened flower buds from both the control and HTS-treated plants were also collected for total RNA extraction. Tissue from the HTS-treated plants was collected in the middle of the light cycle during the fourth day of HTS.
RT-PCR was performed on 128 ng of DNAse-treated total RNA using a One Step RT-PCR kit (Qiagen). Primers and dideoxynucleotide-tailed competimers were used at a 2:4 ratio to amplify a 309 bp segment of 18S rRNA as an internal control for RT-PCR quantification (Sung et al., 2001). Primers specific to B. napus, HSP17.6, HSP70, and HSP101, were designed using sequences obtained from a B. napus EST database held at Agriculture and AgriFood Canada, Saskatoon Research Centre (by courtesy of Andrew Sharpe and Derek Lydiate; e-mail: brassica_est{at}em.agr.ca for further information pertaining to the EST library). Primers were designed to amplify conserved regions of the HSP genes, as determined by alignments with A. thaliana orthologues. The expression patterns of the latter are reasonably well established and, by selecting conserved regions for amplification, it was possible to compare expression patterns between the orthologues of these two closely related species. The Arabidopsis HSP17.6 and HSP101 are heat-inducible, while the B. napus HSP70 shared greatest identity (
85% at the nucleotide level) with constitutively expressed Arabidopsis mitochondrial HSC70–1 (gene location At4g37910). A mitochondrial HSP70 was chosen in order to give some indication of the metabolic impact of the HTS on the high number of mitochondria in the gametophytes. For HSP17.6 and HSP101, the internal 18S rRNA control RT-PCRs were performed using a separate aliquot of the same total RNA. The primers were gene specific and the amplified sequences did not contain intervening introns or intragenic spacers. Equal volumes of HSP17.6 or HSP101 and 18S rRNA reactions were loaded into one well of a 1.5% agarose gel and quantification was performed by comparing HSP band intensities to the 18S rRNA RT-PCR product. Agarose gels were photographed and band intensity determined using a Quantity One GelDoc system (BioRad).
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Results |
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HTS adversely effects B. napus gametophyte function and induces fruit abortion
Quotidian tracking of flowers exposed to HTS allowed in-depth analysis of the effects of HTS on B. napus fertility. HTS did not have an effect on flower production (Fig. 1A), but did have a severe detrimental effect on silique and seed production during the HTS period (Fig. 1B–D). Flower production in 1 week high-temperature-stressed (1WHTS) and 2 week high-temperature-stressed (2WHTS) plants continued at a steady rate throughout the experiment whereas in the control plants, flower production declined to minimal levels after 24 d (Fig. 1). Flowering past 24 d in 1WHTS and 2WHTS plants was due to the production of a significantly higher number of lateral inflorescences (Table 1). The significantly greater number of inflorescences also resulted in an increase in silique and seed production in HTS treated plants later in the experiments, but only after the removal of the HTS treatment (Fig. 1). At 24 d (the end of 2WHTS), all groups of plants had accumulated
6000 flowers (Fig. 1A). In the control group, these flowers developed into 3000 siliques, a 50% conversion, while in the 1WHTS and 2WHTS groups, only 875 (14.6%) and 400 (6.7%) siliques, respectively, developed from these flowers. 1WHTS and 2WHTS reduced flower-to-silique development by 35% and 43%, respectively, compared with that observed in the control population. Over the 25–33 d post-heat-stress flowering period, the rate of silique development by HS plants increased until, in the 1WHTS group, the total number of siliques approached that of the control group while in the 2WHTS group, the total number of siliques produced was 50% of the control group.
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HTS inhibited silique and seed production in those flowers fertilized up to 4 d prior to the initiation of the HTS. On the first day of the HTS (day 10 of flowering), fruit abortion was induced in flowers that had opened on days 9, 8, 7, and 6 of flowering, that is, developing fruit up to 4-d-old were sensitive to HTS, such that an inhibition of fruit production and thus seed production, was observed in flowers from days 6–9 of flowering (Fig. 1).
A decreased level of silique and seed production continued after the removal of the HTS, suggesting that gametophyte function was also adversely affected by HTS (Fig. 1). If HTS-induced flower abortion was the sole reason for decreased fruit and seed production during HTS, then fruit and seed production would have restarted on the same day the HTS was removed. Inhibition of seed production continued for 8 d (1WHTS) and 2 d (2WHTS) after the removal of the HTS stress (Fig. 1C), therefore, it was concluded that gametophyte development and/or function were also adversely affected by the HTS. The 2WHTS plants resumed seed production more quickly than the 1WHTS plants, after removal of the HTS, suggesting that a measure of acclimation to the HTS by the 2WHTS plants occurred during the second week of the HTS.
B. napus inflorescences were unable to acclimatize to the increased temperatures as seed production was inhibited throughout the HTS periods, regardless of the duration of the stress (Fig. 1C, D). After removal of the HTS, the rate of seed production increased rapidly in the HTS treated plants due, in part, to the greater number of lateral inflorescences produced by the HTS treated plants.
HTS limits pollen viability but does not affect microsporogenesis
Two trends were apparent in pollen viability from the HTS treated plants, using FDA staining (Fig. 2). First, overall pollen viability (mean of viability scores from 1 d before opening to 2 d after anthesis) was lower for HTS pollen compared with control pollen after the first 3 d of the HTS period. This decreased level of viability persisted for up to 7 d after removal of the HTS treatment (day 12), but had returned to control levels (81%) by the eighth day after removal of the HTS treatment (day 15).
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The second trend in pollen viability was that 2-d-old flowers exposed to HTS contained a lower percentage of viable pollen (58%), as compared with control flowers of the same age (86%), or –1 or 0-d-old flowers exposed to HTS (78%).
B. napus pollen is trinucleate, so the presence of three nuclei in mature pollen (as observed by DAPI staining) is indicative of the success of microsporogenesis. Pollen that had matured prior to the HTS treatment showed a higher percentage of trinucleate pollen (67%), after 8 h of HTS, compared with that of the control pollen (55%). 74% of pollen collected from freshly opened flowers on the fourth day of the HTS treatment were trinucleated compared with 83% for the control. However, together these data indicate that the pollen reaching maturity under HTS showed no more effects of the HTS on microsporogenesis than that observed under control conditions (Table 2).
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HTS affects in vitro pollen germination and in vitro pollen tube growth
Pollen taken from plants exposed to 4 d of HTS had lower in vitro germination rates (17.5%) than pollen from control-grown plants (59.2%). The lower germination rate for pollen from HTS-reated plants occurred regardless of whether in vitro germination was carried out at 23 °C or 35 °C.
High temperatures during in vitro germination also had a detrimental effect on pollen tube growth. Abnormal pollen tubes were produced by pollen from both the control and 4-d-HTS-treated plants when germinated in vitro at 35 °C, but not at 23 °C (Fig. 3A–D). Pollen tubes from both the control and HTS pollen that developed in vitro at 35 °C for 3 h were thinner and more convoluted than those that developed at 23 °C (Fig. 3C, D). However, pollen tubes from control pollen showed normal morphology when growing towards the micropyle of an ovule in a HTS plant (Fig. 3E).
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Both male and female gametophyte function are adversely affected by HTS
SEM observations revealed that both HTS and control pollen were able to germinate and extend pollen tubes along the septum and towards the micropyles of both HTS or control pistils (Table 3). The ratio of HTS pollen tubes to ovules was lower than that of the control pollen (Table 3); however, the fact that a population of pollen grains from HTS plants successfully germinated in vivo, producing pollen tubes with normal morphology and function suggests that HTS during pollen development results in a reduction in the percentage of pollen able to germinate rather than affecting pollen tube growth and function.
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The results from reciprocal crosses between HTS and control pollen on HTS and control pistils showed that HTS affected both pollen and pistil functionality. Seed-set was reduced by 88% when pollen donor plants were HTS treated and by 37% when the emasculated receptor plants were HTS treated (Table 3). The reduction in silique and seed production in Control pollen x HTS pistil crosses when compared to Control pollen x Control pistil crosses was due to reduced functionality of female gametophytes in the HTS receiver plants. Likewise, the reduction in silique production in HTS pollen x Control pistil crosses was due to reduced pollen functionality.
Some reciprocal crosses were allowed to mature after the removal of the HTS to determine the effect of HTS on fertilization and embryo development (Table 3; Fig. 4). Control pollen x Control pistils produced 19 elongated siliques from 24 crosses (79%), HTS pollen x Control pistils 53%, Control pollen x HTS pistils 68%, and HTS pollen x HTS pistils 8%. The success of seed production was not related to initial silique elongation when one or both parents were HTS treated. While Control pollen x Control pistil crosses did not produce any seedless parthenocarpic siliques, 6%, 3%, and 40% of the elongated siliques were parthenocarpic and seedless in HTS pollen x Control pistil, Control pollen x HTS pistil, and HTS pollen x HTS pistil crosses, respectively. Senescence of the pistil, without elongation, was also quite common if one or both of the parents were HTS treated (Fig. 4, arrows).
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A decrease in seed production was observed as the number of parents treated with HTS increased. Control crosses produced 17.4 seeds per silique compared with 10.9, 3.9, and 0.5 seeds per silique produced by Control pollen x HTS pistil, HTS pollen x Control pistil, and HTS pollen x HTS pistil crosses, respectively (Table 3), indicating that both male and female gametophytes were adversely affected by HTS.
HSP transcription in pollen, pistils, and leaves
B. napus HSP17.6, HSP70, and HSP101 transcription in gametophytic tissues under HTS conditions was determined using RT-PCR amplification from total RNA. It had previously been reported that the presence of HSPs improved tissue thermotolerance, therefore, determining HSP gene expression patterns may help explain why differential thermotolerance was observed between male and female gametophytes.
HSP17.6 transcripts were detected only in pollen from HTS-treated plants (Fig. 5A). No expression was observed in mature, control pollen, control and HTS pistils or control and HTS leaves.
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HSP70 transcripts were not detected in pollen, but were present in control and HTS-treated pistils and leaves (Fig. 5B). HTS did not appear to increase HSP70 expression in either pistils or leaves.
HSP101 transcripts were detected in all tissue types except mature pollen from control plants (Fig. 5C). HTS pistils had a higher amount of HSP101 transcript, compared with control pistils. By contrast, no increase in HSP101 mRNA in HTS leaves, compared with control leaves, was observed.
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Discussion |
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Fruit and seed development are co-ordinated processes that are initiated after fertilization (Gillaspy et al., 1993). In B. napus DH12075, HTS reduced seed production by almost totally inhibiting or preventing fruit development during the HTS period. Reduced micro- and megagametophyte fertility combined with a failure of post-fertilization events resulted in an almost total abolition of seed production during the HTS period.
Reduced seed-set in HTS-treated plants was not a consequence of reduced pollen viability. Reduced pollen germinability was probably a major contributor to reduced HTS pollen fertility. Pollen developing under HTS conditions had lower in vitro germination rates at 23 °C than control pollen under the same conditions. Pollen germinating in vitro at 35 °C for 3 h had thinner pollen tubes with stunted, convoluted morphology (Fig. 3C, D). Similarly, pollen from both L. longiflorum (lily) and N. tabacum (tobacco), germinated in vitro under HTS conditions (39 °C for 30 min), showed stunted pollen tube morphology. However, this phenotype was reversible on removal of the HTS (Herpen et al., 1989). In vitro germination rates, at both 23 °C and 35 °C, of B. napus HTS pollen were at similar low rates suggesting that HTS-induced changes in B. napus pollen development are irreversible. If the effects of HTS on B. napus pollen were reversible, then fruit and seed production during the 1WHTS or 2WHTS, due to normal pollen function during the cooler night-time periods, would have been seen. No fruit and seed production was observed during the HTS periods.
It has recently been established that successful fertilization requires the maternal guidance of the pollen tube through the maternal sporophytic tissue, to the embryo sac (Higashiyama et al., 2001; Rotman et al., 2003). The guiding molecule is thought to be diffusible and produced from the synergid cells of the egg sac (Higashiyama et al., 2001, 2003; Huck et al., 2003; Rotman et al., 2003). In B. napus, the effects of HTS on pollen tube growth, through the pistil to the micropyle, appeared to be minimized by the surrounding maternal tissue. Both HTS-developed pollen on control stigmas and control pollen on HTS-treated stigmas, were able to germinate and produce tubes that were successfully guided to the micropyle of the ovule. However, it was not possible to show penetration of the micropyle by the pollen tube. These results are contrary to those of Higashiyama et al. (2003) who demonstrated in Torenia fournieri, that HTS-treated ovules were unable to attract pollen tubes. In Brassica napus, it appears that HTS does not disrupt the synergid-derived, pollen tube guidance system to the micropyle.
In both B. napus and L. esculentum, it appears that microspore and/or pollen development are sensitive to HTS (this work; Peet et al., 1998; Sato et al., 2002), while mature pollen appears to respond to HTS in a species-dependent manner. In this study, mature B. napus pollen showed only a small reduction in viability when HTS treated, whereas mature Z. mays pollen grains showed a severe decrease in germinability after a 38 °C HTS for 24 h (Herrero and Johnson, 1980). By contrast, B. juncea pollen grains were still able to germinate after 4 or 24 h at 45 °C or 60 °C (Rao et al., 1992) and N. sylvestris (Speg) and Petunia hybrida (Vilm) pollen germination frequencies were not affected by temperatures of up to 60 °C for 48 h (Rao et al., 1995). Differences in thermosensitivity of mature pollen from monocotyledonous and dicotyledonous plants may exist, however, further studies are required before conclusions can be drawn.
The ability of mature B. napus pollen to tolerate HTS does not appear to be the result of increased HSP synthesis as determined from a subset of HSP transcripts. Mature B. napus pollen, heat-shocked for 30 or 60 min at 35 °C was unable to synthesize a subset of HSP transcripts (Fig. 5). The already desiccated nature of mature B. napus pollen probably protected it from damage by high temperatures, making it unnecessary to up-regulate HSP transcription for thermoprotection.
On the other hand, both HSP17.6 and HSP101 transcripts were induced in B. napus pollen developing under HTS conditions, suggesting that developing microspores are responsive to HTS. In Z. mays pollen developing under HTS conditions, elevated levels of small HSPs, above those observed in non-HTS pollen (Dupuis and Dumas, 1990) and HSP101 mRNA and protein (Young et al., 2001), have been reported.
While some HSP70 transcripts have been observed in developing microspores of both L. esculentum and Z. mays under control or HTS conditions, they were not detected in mature pollen (Duck and Folk, 1994; Gagliardi et al., 1995). By contrast, mitochondrial HSP70 transcripts were not detected in B. napus microspores that had developed under HTS conditions although they were detected in control and HTS leaves (Fig. 5). Similarly, the Arabidopsis orthologue (MTHSC70-1) is not up-regulated in vegetative tissue by HTS although it is induced during seed germination (Sung et al., 2001). Expression of MTHSC70-1 was not determined in Arabidopsis pollen. These observations suggest that the B. napus HSP70 gene, orthologous to the Arabidopsis MTHSC70-1 gene, is regulated in a tissue-specific, non-HTS inducible manner. Due to the plants being well-watered throughout the experiment, transpiration cooling effects may have contributed to the lack of HSP mRNA induction in HTS-treated leaves.
The reciprocal Control/HTS pollen x Control/HTS pistil crosses show that megagametophyte fertility was affected by HTS. A susceptibility of the megagametophyte to HTS has been previously suggested in B. napus (Polowick and Sawhney, 1988; Saini et al., 1983), T. aestivum (Saini et al., 1983), and L. esculentum (Peet et al., 1998; Sato et al., 2002). By contrast, in HTS-treated Z. mays, the megagametophytes appear to be unaffected and it is desiccation and loss of pollen viability that are thought to be the primary cause of heat-induced yield reductions (Herrero and Johnson, 1980; Dupuis and Dumas, 1990).
HTS effects on both gametophytes appeared to be synergistic, such that when both were HTS treated, fewer fruit and seeds were produced than was expected from the additive effects of single HTS-induced gametophyte dysfunction (Table 3). Conversely, the interaction of a non-stressed gametophyte with its opposite stressed partner may have been sufficient to rescue a measure of combined gametophyte function, such that a significant number of fruit and seed were produced in these hetero-stressed systems. Similar results have been reported in tomato (Peet et al., 1998) and wheat (Saini et al., 1983), where it also appears that HTS induced sterility is due to a synergistic combination of both micro- and megagametophyte dysfunction.
A lack of fruit and seed production from flowers fertilized up to 4 d prior to the onset of HTS suggests that, as well as affecting micro- and megagametophyte function, HTS disrupted post-fertilization events, resulting in the cessation of development of the fertilized megagametophytes/carpels into seed bearing siliques (Fig. 1C). If a disruption in megagametophyte/carpel development was the sole effect of HTS, then seed-bearing siliques should have been produced immediately after the removal of the HTS. This was not the case, therefore it is concluded that gametophyte sterility, together with disruptions in carpel development, were responsible for fruit abortion and reduced seed set in B. napus under HTS conditions. The production of seedless, parthenocarpic siliques, when both parents were HTS-treated (Fig. 4), suggests that pollination (or the events immediately following) initiates fruit formation and that HTS in B. napus can induce the uncoupling of fruit formation from seed development. That parthenocarpic siliques were much shorter than fully developed siliques was possibly due to low levels of endogenous growth hormones resulting from an absence of seeds (Vivian-Smith and Koltunow, 1999). HTS-induced production of parthenocarpic fruit has been reported in Capsicum annuum (Erickson and Markhart, 2002), B. juncea (Rao et al., 1992), and L. esculentum (Peet et al., 1998; Sato et al., 2001).
In this study, the effects of HTS on fruit and seed production in B. napus have been described. It was observed that HTS had no effect on flowering, but did induce flower abortion and parthenocarpic silique production. Analysis of micro- and megagametophyte function indicated that maternal guidance of pollen tube germination and growth was maintained during HTS, however, possible impacts on micropyle penetration, fertilization and post-fertilization cannot be ruled out. It would be possible to investigate such impacts by microarray analysis or by screening for appropriate lack of post-fertilization silique development mutants in Arabidopsis. A basis for such a screen would be similar to those conducted for mutants exhibiting seedless parthenocarpic silique production, for example, fruit without fertilization (Vivian-Smith et al., 2001). This approach would identify the molecular lesion(s) responsible for the HTS-induced disruptions in fruit and seed production in the Brassicaceae.
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We would like to thank Fatma Kaplan from Charles Guy’s laboratory (University of Florida) for designing and testing the 18S rRNA RT-PCR internal control primers and Andrew Sharpe and Derek Lydiate, Agriculture and AgriFood Canada, Saskatoon Research Centre for allowing us to search the B. napus EST database for the B. napus HSP sequences. Funding for this research was through the Canola Council of Canada, the Saskatchewan Canola Development Commission, and NSERC, Canada.
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