高温导致绒毡层中基因表达的变化引起不育

Plant male reproductive development is highly organized and sensitive to various environmental stressors, including high temperature. We have established an experimental procedure to evaluate high temperature injury in japonica rice plants. High temperature treatment (39 ° C/30 ° C) starting at the microspore stage repeatedly reduced spikelet fertility in our system. Morphological observations revealed that pollen viability in plants exposed to high temperatures was lower than that in control plants. Most pollen grains in high temperature-treated plants displayed a normal round shape and stained reddish purple with Alexander’s reagent; however, the pollen grains were very poorly attached and displayed limited germination on the stigma. To investigate gene regulatory mechanisms in the anther in high temperature environments, DNA microarray analysis was performed by comparing non-treated samples with samples treated with 2–4 d of high heat. Genes responsive to high temperatures were identifi ed from clustering of microarray data. Among these, at least 13 were designated as high temperature-repressed genes in the anther. Expression analyses revealed that these genes were expressed specifi cally in the immature anther mainly in the tapetum at the microspore stage and down-regulated after 1 d of high temperature. The expression levels of Osc6 , OsRAFTIN and TDR , which are tapetum-specifi c genes, were unaffected by high temperatures. These results suggest that not all tapetal genes are inhibited by increased temperatures and the tapetum itself is not degraded in such an environment. However, high temperatures may disrupt some of the tapetum functions required for pollen adhesion and germination on the stigma. Keywords: High temperature stress • Male sterility • Microarray • Rice • Tapetum . Abbreviations : AD , auricle distance ; GO , gene ontology ; RT–PCR , reverse transcription–PCR. Introduction Rice ( Oryza sativa L.) is one of the world’s most important cereals; however, grain yields often fl uctuate due to various environmental stressors. Temperature during reproductive development is an important factor determining grain yields in rice ( Satake and Yoshida 1978 , Nishiyama 1984 , Prasad et al. 2006 ). To date, many studies have focused on low tem- perature-induced pollen sterility in rice ( Nishiyama 1984 , Wen et al. 2002 , Oliver et al. 2005 ) because, compared with other cereal crops, such as wheat ( Triticum aestivum ) and barley ( Hordeum vulgare ), rice is much more sensitive to low temperature due to its tropical origin. However, global climate change is likely to exacerbate current crop vulnera- bility to environmental stress. In particular, the sexual repro- ductive phase is predicted to be vulnerable to the effects of global warming ( Hedhly et al. 2009 ). Global warming may increase the instability of rice yields even in temperate regions, mainly due to the increased probability of male ste- rility induced by high temperatures ( Horie et al. 1996 ). Male reproductive development in higher plants is known to be very sensitive to abiotic stress. In particular, high or low temperature stress results in a lower seed set due to male sterility in most crops, including tomatoes ( Peet et al. 1998 , Sato et al. 2002 ), cowpeas ( Ahmed et al. 1992 ), wheat ( Saini et al. 1984 ), barley ( Sakata et al. 2000 , Koike et al. 2003 , High Temperatures Cause Male Sterility in Rice Plants with Transcriptional Alterations During Pollen Development Makoto Endo 1 , 6 , Tohru Tsuchiya 2 , Kazuki Hamada 3 , Shingo Kawamura 3 , Kentaro Yano 3 , Masahiro Ohshima 1 , Atsushi Higashitani 4 , 5 , Masao Watanabe 4 , 5 and Makiko Kawagishi-Kobayashi 1 , ∗ 1 Rice Biotechnology Research Team, National Institute of Crop Science, NARO, Tsukuba, Ibaraki, 305-8518 Japan 2 Life Science Research Center, Mie University, Tsu, 514-8507 Japan 3 Faculty of Agriculture, Meiji University, Kawasaki, 214-8571 Japan 4 Graduate School of Life Sciences, Tohoku University, Katahira, Sendai, 980-8577 Japan 5 Faculty of Science, Tohoku University, Aramaki-Aoba, Sendai, 980-8578 Japan Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org 6 Present address: Takii Plant Breeding Experiment Station, Konan, Shiga, 520-3231 Japan. ∗ Corresponding author: E-mail, makikokk@affrc.go.jp ; Fax, + 81-29-838-8484 . 1911Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009. Regular Paper at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from Oshino et al. 2007 ) and rice ( Satake and Yoshida 1978 , Nishiyama 1984 , Prasad et al. 2006 ). In fl owering plants, male gametogenesis from archesporal cells to mature pollen involves a series of complicated events ( Goldberg et al. 1993 ). During male reproductive development, the young microspore stage is known to be highly sensitive to environ- mental stress, and cold stress in rice is particularly well known. The young microspore stage during or just after microspore release from the tetrad is the most vulnerable to low temperatures ( Nishiyama 1984 , Oliver et al. 2005 ). At this stage, the tapetum, the innermost cell layer of the anther locule, is thought to be fully developed and actively providing nutrients, components and enzymes for microspore development. It is known that the tapetum is severely damaged in rice plants exposed to low temperatures at the young microspore stage ( Nishiyama 1984 ). Satake and Yoshida (1978) reported that exposure to high temperatures induced sterility, and rice plants were most sensitive to excessive heat during two periods, namely the fl owering stage and the young microspore stage. In high temperature injury during fl owering, spikelet sterility is mainly due to poor anther dehiscence ( Satake and Yoshida 1978 , Matsui et al. 1997 ). Furthermore, it has been reported that high temperatures on the day of fl owering decrease the ability of pollen grains to swell, thus resulting in poor anther dehiscence ( Matsui et al. 2000 ). However, little is known about how high temperatures at the microspore stage induce sterility in rice. In order to gain insight into the mechanisms of sterility induced by high temperatures at the microspore stage in rice, we observed pollen structure and germination after high temperature treatment and analyzed the relative gene expression levels during treat- ment each day. Results High temperature treatment at the microspore stage reduced spikelet fertility To investigate the effects of high temperatures on spikelet fertility, rice plants were exposed to temperatures of 39 ° C in the daytime and 30 ° C at night for 7 d at three different developmental stages. The stages were early panicle devel- opment, glumous fl ower primordium differentiation and the early microspore stage following meiosis. The results clearly indicated that the microspore stage was the most sensitive to heat stress, and spikelet fertility was completely lost at this point ( Table 1 ). This fi nding was consistent with a previous study reporting that reduction of spikelet fertility in rice was caused by high temperatures during the booting stage, 9 d before heading ( Satake and Yoshida 1978 ). Since microsporogenesis is known to be highly sensitive to various stresses, we performed a detailed analysis focused on the effects of high temperatures at the microspore stage. The developmental stages were estimated by auricle distance (AD), which is the distance between the auricle of the fl ag leaf and that of the penultimate leaf. High temperature treatments were started at AD = 0 cm, corresponding to the early microspore stage. Because 5 d of treatment resulted in complete sterility, we examined the effects of a shorter expo- sure to high temperatures. Rice plants were exposed to high temperatures (39 ° C/30 ° C) for 2–4 d, and were subsequently transferred back to a normal environment (28 ° C/22 ° C) until seed maturity ( Fig. 1 ). Spikelet fertility drastically decreased when the plants were treated with high temperatures for 2–4 d ( Table 2 ). Longer exposure showed more severe effects on spikelet fertility, and the 1 d treatment resulted in only a limited effect. These results suggest that heat stress at the microspore stage for 2 d was suffi cient to induce spikelet sterility and irreversibly damage some aspects of panicle development. Pollen grains exposed to high temperatures at the microspore stage displayed limited germination on the stigma To gain insight into how high temperatures reduce spikelet fertility, the anther, pollen and pistil were carefully observed through a microscope. At heading, the anther and pistil of the high temperature-treated plants were indistinguishable from those of the untreated plants in shape and size (data not shown). Pollen viability of untreated and plants and plants treated for 2 or 4 d was tested using Alexander’s reagent ( Alexander 1969 ). As shown in Fig. 2A , high tem- perature treatments increased the number of spikelets con- taining inviable pollen, while all 36 spikelets showed ≥ 75 % pollen viability in the untreated plants. However, 28 and 33 spikelets out of 36 showed ≥ 50 % pollen viability in the plants treated for 2 or 4 d, respectively ( Fig. 2A ). The spikelets in high temperature-treated plants had a signifi cant amount of pollen grains which stained reddish purple ( Fig. 2B ). We observed a notable difference in the numbers of pollen grains on the stigma between the high temperature-treated and untreated plants. In the untreated plants, we observed Table 1 Spikelet fertiltiy of rice plants after exposure to high temperature at different developmental stages Treatment Spikelet fertillity ( % ) High temperature I 60.9 II 59.8 III 0.0 Untreated 78.6 Rice plants were exposed to high temperature at developmental stages estimated as I, early panicle development; II, glumous fl ower primordium differentiation; III, early microspore following meiosis. 1912 M. Endo et al. Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from vigorous pollen germination and pollen tube elongation on the stigma after pollination ( Fig. 3A ). Conversely, the number of pollen grains on the stigma in the high temperature- treated plants was drastically reduced and the pollen on the stigma had barely germinated ( Fig. 3B ). No difference was detected between the high temperature-treated and untreated plants in regard to the opening of the spikelets or anther dehiscence at fl owering (data not shown). It is possi- ble that most of the pollen grains did not adhere to the stigma or were washed off during the staining process due to poor germination. We subsequently performed crosses using high temperature-treated plants as female parents to inves- tigate the effects of heat stress on the pistil. Pollen from untreated plants was shed onto the high temperature- treated pistil, and thereafter the pollen germination was observed after staining with aniline blue. The normal extent of pollen germination and pollen tube elongation was observed in 20 out of 24 crosses pollinating high temperature-treated pistils with untreated pollen, and then the progeny from these crosses grew normally. Representative results are shown in Fig. 3C . The number of pollen grains on the high temperature-treated stigma was similar to that observed on the untreated stigma ( Fig. 3A, C ). These results suggest that high temperatures at the microspore stage did not damage the ability of the pistil to receive pollen by the stigma, but impaired the ability of pollen to attach and/or germinate on the stigma. Identifi cation of high temperature-responsive genes in the anther using DNA microarray To gain insight into the molecular mechanisms of heat- induced male sterility, we analyzed transcriptional altera- tions in the anther under high temperature conditions using a 22K rice oligo DNA microarray (Agilent Technologies, Palo Alto, CA, USA). Total RNA was extracted from the anthers exposed to high temperatures for 2, 3 and 4 d (H2, H3 and H4) and from untreated anthers with the corre- sponding timing (C2, C3 amd C4). RNA was also prepared from seedlings exposed to high temperatures for 4 d (HS) and from seedlings without treatment (CS) to distinguish AD -1~1 cm Co nt ro l H ig h te m pe ra tu re tre at m en t 2 days 3 days 4 days C2 C3 C4 H2 H3 H4 Heading CM H2M H4M Fig. 1 A schematic illustration of the high temperature treatment. Rice plants were grown in a greenhouse at 28 ° C in the daytime and at 22 ° C at night (28 ° C/22 ° C) as a normal condition (thin arrows). The high temperature treatment started at auricle distance (AD) = −1 to + 1 cm, corresponding to the early microspore stage. During the high temperature treatment, plants were cultivated in a growth chamber at 39 ° C/30 ° C (thick arrows). RNA was extracted from the anthers after high temperature treatment from day 2 to day 4 (H2, H3 and H4) and from the corresponding untreated anthers (C2, C3 and C4). Some of the high temperature-treated and untreated plants were grown at 28 ° C/22 ° C until maturity (H2M, H4M and CM) in order to analyze pollen viability and seed fertility. 0 5 10 15 20 25 30 35 40 75–100 50–75 25–50 0–25 Pollen viability of each spikelet (%) N um be r o f s pi ke le ts CM H2M H4M A B CM H2M H4M Fig. 2 Viability of the high temperature-treated pollen. Anthers were harvested just prior to anthesis and subjected to Alexander’s staining. (A) Pollen viability was expressed as a percentage of the stained pollen. The viability of pollen from 36 spikelets was scored for each plant. (B) Typical anthers stained with Alexander’s reagent. CM, untreated; H2M and H4M, high temperature treated for 2 or 4 d, respectively. Bars = 0.1 mm. Table 2 Spikelet fertility of high temperature-treated plants Experiment High temperature-treated Untreated 1 d 2 d 3 d 4 d 1 – a 5.3 0.6 3.9 71.5 2 – 29.8 8.5 8.3 61.0 3 – 17.8 20.4 9.7 81.2 4 63.9 24.5 – 5.5 85.1 a Data not available. 1913 Rice male sterility due to high temperatures Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009. at China Academ y of Agricultural Sciences on M arch 20, 2010 http://pcp.oxfordjournals.org D ow nloaded from the specifi c transcriptional alteration along with anther development from the general heat shock response in the vegetative organs. These samples analyzed by DNA microarray are schematically shown in Fig. 1 . To screen high temperature- responsive genes, we initially chose 1,439 genes that show a > 2-fold difference of expression in either one of the three comparisons, H2/C2, H3/C3 or H4/C4. These selected genes were subsequently classifi ed into 30 clusters according to their expression pattern ( Fig. 4 ). In clusters 2 and 15, the genes were down-regulated by high temperature treatments specifi cally in the anther after just 2 d. Clusters 10 and 29 contained genes whose expression gradually decreased during the high temperature treatment. Clusters 4, 6 and 30 contained genes that were highly up-regulated after high temperature treatment for 4 d. The genes belonging to each cluster are listed in Supplementary Table S1 and the gene classifi cation based on the gene ontology (GO) in 30 clusters is shown in Supplementary Fig. S1. We focused on the genes down-regulated by high tem- peratures, because it is assumed that the genes important for pollen development and fertility were impaired under high temperature conditions. The gene expression in cluster 2 and cluster 15 (containing 15 and 56 genes, respectively) was severely impaired after only 2 d but it was not greatly affected after 3 or 4 d of high temperatures. We noted these genes showing a drastic decrease after 2 d of high tempera- tures, because the response to 4 d of high temperatures could have resulted from various indirect effects and 2 d of heat was suffi cient to induce sterility. According to the GO classifi cation, some of the genes in clusters 2 and 15 were presumably involved in the metabolic process and related to the catalytic activities (Supplementary Figure 1). We chose all 15 genes in cluster 2 and three genes from cluster 15 (listed in Table 3 ) for the individual gene expression analysis. The relative expression levels of these 18 selected genes were analyzed by real-time reverse transcription–PCR (RT–PCR) using RNA samples from two independent heat stress experiments, one of which was the same as used for a DNA microarray analysis. Among the selected 18 genes, we analyzed the relative gene expression levels of 16 genes, because the PCR primers for two genes, AK102387 and AK073529, did not work. In 15 out of 16 genes tested, real-time RT–PCR analysis demonstrated the drastic down- regulation due to high temperatures that had been observed in microarray analysis. The relative expression levels of these 15 genes are shown in Fig. 5 . A common feature of these genes (except for AK062288 and AK058903) was that expres- sion was seen until day 2 without treatment and was drasti- cally impaired within 1 d of the high temperature treatments. These 13 genes were therefore designated as high temperature- repressed genes. Tissue specifi city of the genes that were immediately down-regulated in response to the high temperature treatment Among the high temperature-repressed genes we identifi ed YY1 and YY2 as immature anther-specifi c genes which are mainly expressed in the rice tapetum ( Hihara et al. 1996 ). The highest expression of YY1 and YY2 (AK107918 and AK105510, respectively) in non-treated anthers was observed on days 0 and 1, which correspond to the microspore stage ( Fig. 5 ). This fi nding was consistent with the previous report by Hihara et al. (1996) . Because the temporal expression profi le was shared among 13 genes, including YY1 and YY2 , we examined whether these genes were expressed in the same tissue. The expression of genes shown in Fig. 5 was analyzed by real-time RT-PCR using various tissues. As expected, high temperature-repressed genes were highly expressed in the immature anther but were not detected in the root, leaf, pistil, callus or mature anther ( Table 4 ). Furthermore, a detailed expression analysis of these genes revealed that the highest level of expression was in anthers with a fully developed tapetum at the microspore stage ( Fig. 6 ). To investigate where these high temperature-repressed genes A B C Fig. 3 Pollen germination on stigma. Aniline blue staining of a self-pollinated stigma of an untreated plant (A), a self-pollinated stigma of a plant exposed to high temperature for 4 d (B) and a cross-pollinated stigma of a plant exposed to high temperature for 4 d with untreated pollen (C). Bars = 0.1 mm. 1914 M. Endo et al. Plant Cell Physiol. 50(11): 1911–1922 (2009) doi: