Development of a new heat tolerance assay system for rice spikelet sterility
© The Author(s) 2017
Received: 18 August 2016
Accepted: 2 May 2017
Published: 10 May 2017
Reduction in rice yield caused by high temperature-induced spikelet sterility has been a serious concern in rice production. To date, several screening methods have been used, although their reproducibility is sometimes poor due to artifacts mainly caused by varietal differences in heading dates and panicle heights (i.e., the distance from the lamps).
We have developed a novel assay system for heat-induced spikelet sterility by using artificial rice paddies in phytotrons to conduct a highly reproducible assay throughout a year. Plants restricted to the main culm were treated under a series of heat conditions, and height uniformity of each plant was ensured by using height-adjustable pots.
Results suggested that a 3-day heat treatment of 35 °C-day/29 °C-night cycles was the most suitable condition. Under the treatment, two distinct groups were identified among nine heat tolerant cultivars, with no varietal difference in panicle temperature, indicating that the system is capable of eliminating the varietal difference in panicle temperature.
It is concluded that the assay system would be a powerful tool for selecting heat tolerant varieties, as well as the analysis of genetic factors from various cultivars, eliminating potential artifacts.
At present, deterioration of rice quality caused by heat stress during ripening has been a serious concern in rice production owing to climate change [1–3]. However, as air temperatures increase, spikelet sterility caused by high temperatures is also expected to become a serious problem in rice production globally, including Japan, as simulated by Horie et al. . In fact, severe heat-induced rice spikelet sterility caused by high temperatures during flowering was observed in the summer of 2003 in China . As global warming advances, the instability of rice production will be enhanced owing to the increased frequency of heat-induced spikelet sterility .
Spikelet sterility caused by high temperatures is known to occur following heat exposure of panicles during booting and flowering . It begins to occur when the daily maximum temperature reaches approximately 34–36 °C [8–10], and high humidity promotes spikelet sterility [10, 11]. Low humidity [8, 11], wind , and high CO2 concentration  also influence sterility. The mechanism(s) of rice spikelet sterility involves the lack of full development of pollen  and faulty dehiscence of anthers [7, 13] caused by high temperatures, resulting in a reduction in the number of healthy pollen grains at the stigma . Consequently, heat-induced spikelet sterility causes a decline in fertility. However, the exact mechanism(s) of and the genes involved in heat-induced spikelet sterility remain unclear.
The impact on rice yield in Japan following further global warming using a crop simulation model suggested that the increasing high temperature tolerance by approximately 1.5 °C during the flowering period would greatly reduce the incidence of spikelet sterility [4, 14]. Therefore, the promotion of rice breeding of heat tolerant varieties is urgent, as well as understanding the mechanism(s) of high temperature-induced spikelet sterility, and the development of future adaptation technologies. To date, varietal differences in heat tolerance of spikelet sterility have been reported [9, 11, 13, 15–19]. The reported tolerant cultivars include three Indica cultivars, IR36, IR24, and IR64, two Japonica cultivars, Akitakomachi and Koshihikari, and an aus cultivar, N22. These cultivars were selected using three methods conducted under variable conditions, including temperature gradient chambers (TGC) under natural light, phytotrons, and greenhouses. To our knowledge, there has, to date, been no direct comparison of heat tolerance among these cultivars, presumably due to the technical limitations of each method, availability of germplasm, and expected growth response differences of indica and japonica types under specific screening conditions. Additionally, the optimal conditions for assaying heat-induced spikelet sterility have not been established.
It is generally known that panicles are the rice plant’s heat-sensitive organ . When evaluating cultivars in phytotron assays, one concern was that cultivar-to-cultivar variations of the panicle position potentially exist, which often causes variations in the distances between lamps and panicles. To reproducibly test heat-induced spikelet sterility, we have developed a new custom-built assay system that can be performed in two environmentally controlled growth chambers. In this system, the panicle top position in the artificial paddies that simulate the environmental conditions of the rice paddy (referred to as ‘an artificial rice paddy’) have been made to be adjustable, so that the position of all cultivars/lines can be uniform. We report here the robustness and reproducibility of the assay system by testing both high temperature-induced spikelet sterility and panicle temperature among the reported heat-tolerant rice cultivars.
Plant materials and growth conditions
Rice cultivars and genotypes used in this study
Heat stress assay system in a phytotron
High temperature treatment
On heading day, the potted plants were transferred to another phytotron, and exposed to cycles of 30–36 °C (13 h light)/26–30 °C (11 h dark) at 60% RH for 3 days. The height of the panicle tip was maintained at 70 cm by adjusting the pot from the water surface of the artificial rice paddy each day. For N22, which had a relatively large panicle height, the pot was set to the lowest position, and in case the height became >70 cm the culm was gently leaned over the adjacent pot space to ensure the same panicle tip height (70 cm above the water surface of the artificial rice paddy). The treated plants were then transferred to the previous phytotron and exposed to 26 °C (13 h light)/22 °C (11 h dark) at 60% RH until harvest.
Measurement of panicle temperature
According to Maruyama et al. , a data logger system (CR23X; Campbell Scientific, Inc., USA) was used to measure the spikelet temperature (referred to as ‘panicle temperature’) for one day after inserting a fine wire copper-constantan thermocouple into a spikelet between glumes on the second day of heat treatment. The spikelet was measured at approximately one third of the distance from the tip of the panicle.
Assay of spikelet fertility
After harvest, the panicles were dried at 30 °C for 3 days, separated into three sections (upper, middle, and under sections) in the primary rachis branches, and the number of mature seeds and empty caryopses of panicles of the middle section were counted.
Data were subjected to analysis of variance (ANOVA) using JMP software (version 12.1.0, SAS Institute Inc., Cary, NC). Analysis of the spikelet fertility and panicle temperature data was conducted using a Tukey’s Honestly Significant Difference (HSD) test with JMP software.
Design of a heat stress assay system in a phytotron
Determination of optimized conditions for identifying varietal differences
Varietal difference of nine heat tolerance cultivars
In this study, we have developed a reliable screening system performable in a phytotron for high temperature-induced spikelet sterility in rice (Fig. 1). In this system, providing only the main culm and fixing the distance from the lamp to the panicle allowed us to rule out the potential artifact of cultivar-to-cultivar variations in lamp-to-panicle distances. Consequently, it has been demonstrated that our system was capable for identifying two distinct tolerant groups even among nine tolerant cultivars previously reported.
The robustness of the developed high temperature-induced spikelet sterility assay system
Consistent with Maruyama et al. , we also observed similar varietal differences obtained in the TGC that are required to generate the temperature-versus-fertility curve in each cultivar. Hence, it was concluded that our system is capable of simply but reproducibly evaluating varietal differences of high temperature-induced spikelet sterility without generating any curve in each cultivar. Unlike leaves, the panicle is likely to be a heat-sensitive organ , and thus the distance from lamp to panicle should be a crucial factor influencing spikelet fertility. Therefore, it was essential to exclude the potential artifact by keeping the distance between lamps and panicle tips uniform when evaluating potential varietal differences under the artificial light in a phytotron. In the preliminary experiment, ten plants per 1/5000 Wagner pot were transplanted and grown “without removing tillers”, which provided considerably various panicle heights even for the same cultivar in a pot. When the same eight cultivars were examined by fixing the distance between the lamp and the panicle (Additional file 2: Fig. S2A), the evaluation failed. The trend obtained in the preliminary evaluation differed from Maruyama et al.  and the results of this work in which AKT, the high tolerance cultivar, had the lowest spikelet fertility, whereas conversely the sensitive cultivar NKM had comparatively high tolerance (Additional file 2: Fig. S2B). This contradiction could be explained by the variation in panicle heights in each pot. Additionally, the treatment of an individual plant at heading was unlikely to be uniform, presumably because of the differences in heading dates of each panicle in one pot, together with the difference in panicle heights among 10 individuals. These issues could be solved by using the newly designed pots with a height adjustment function (see Fig. 1) in the artificial rice paddy even with another potential influence of panicle excursion rate (typically, between 2 and 5 cm/day, see ) (Figs. 1, 2). It is true that the optimization of the adjustable height system may be another issue to address prior to the experiment. Although the proposed height adjustment in each cultivar was successfully conducted for three days during the examination and did not require any further adjustment, except for N22 (see “Methods”), it may be necessary to extend the size of height-adjustment, depending on the cultivars.
Maruyama et al.  reported eight varietal differences using a temperature gradient chamber (TGC). Their system measured spikelet sterility under various temperature conditions that included a temperature gradient in the chamber, resulting in the detection of varietal differences to heat exposure with accuracy. However, there was a technical limitation for the number of cultivars to be tested, and under natural light, the assay could be influenced by fluctuating environmental conditions (e.g., shade). Additionally, this method requires more time and effort in checking panicle temperature, in addition to the difficulty of temperature management throughout a whole year. In contrast, our phytotron system, presented here, requires less effort and enables a reliable evaluation of heat tolerance among a greater number of candidate cultivars under fixed temperature conditions throughout a year.
Comparison with tolerance evaluation of previous reports
We revisited the question of whether the results obtained in the indirect TGC method hold true. Tolerance of heat-induced spikelet sterility in eight cultivars including 6 japonica and 2 indica cultivars used in Maruyama et al.  was determined by the temperature (T 75) at which spikelet fertility becomes 75% of that of the control plants. They reported tolerance to spikelet sterility under high temperature was in the order of highest to lowest of; IR36, IR24, AKT, KSH, HTM, NPB, HNH, and NKM. In our study, following the 35 °C/29 °C treatment the tolerance to spikelet sterility was in the order of highest to lowest of; IR36, IR24, AKT, HTM, NPB, NKM, KSH, and HNH, and indicated similar varietal differences to those seen previously, except for the tolerance of KSH (Fig. 4A). They were shown to correlate (R = 0.77, p < 0.05). Several investigators showed that an aus cultivar, N22, had very high heat-induced spikelet sterility tolerance [15, 17, 18]. Other tolerant cultivars reported include NPB, AKT [9, 13], and KSH . We evaluated all of these tolerant cultivars at once under high temperature conditions using the current system. Our results showed that the tolerance of N22 was prone to be higher than IR36, and the other cultivars showed similar varietal differences among the eight cultivars used by Maruyama et al.  (Fig. 4A), suggesting that this spikelet sterility assay system had high reliability and could be used to identify varietal differences even among cultivars (Figs. 3, 4) in the phytotron as an alternative method of TGC. Further evaluations using other candidate cultivars with high heat tolerance will be of importance in relation to breeding.
Furthermore, it was necessary to identify cultivars with heat tolerance greater than N22 for breeding purposes. The data suggested that the 3-day 35 °C/29 °C treatment allowed us to detect varietal differences even when the tolerance level indicated a numerical value above the 65.1% spikelet fertility of highly tolerant N22 cultivar (Fig. 3). Selecting highly heat tolerant cultivars from our genetic resources is currently under investigation.
Panicle temperature displays varietal differences
Panicle temperature based on T 75 showed the certain varietal differences from +0.1 °C to −2.5 °C treatment temperatures . In our phytotron assay system, the position of each potted plant was randomized in the artificial rice paddy to minimize possible variations in the set air temperature. The panicle temperature observed in the 35 °C/29 °C treatment showed that the recorded panicle temperatures of all cultivars were slightly higher than the air temperature (Fig. 4C). This difference might be explained by the absorption of the infrared rays from the metal halide lamps in panicles, as Yoshimoto et al.  discussed. Time course data during the period from insertion to removal of the sensors indicated relatively stable panicle temperature on the second day even with dynamic changes in water temperature (Additional file 1: Fig. S1), which is consistent with Nishiyama and Satake  who reported that the panicle was sensitive to heat. Under the field conditions, certain varietal differences in panicle temperature by evapotranspiration of the community or in panicle height are known to be important characters for high temperature-induced spikelet sterility tolerance . Therefore, one would expect that the 1.2 °C difference might be due to the varietal differences of panicle temperatures; however, it should not be ignored that there was essentially no statistical difference in panicle temperature among cultivars, but there was a clear varietal difference for spikelet fertility, as shown in Fig. 4. Moreover, we did not observe any clear correlation between spikelet sterility tolerance and panicle temperature in this study. This has implied another possibility that high-temperature induced spikelet sterility may be superimposed by several factors including panicle temperature. Our data also suggest that the newly developed system is capable of isolating the influence of panicle temperature. Such a reproducible tolerance assay that could similarly eliminate potential artifacts will be useful in future studies. This may directly lead to the discovery of metabolites or genes that participate in the cause organization of spikelet sterility like the anther or pollen.
A list of the main components of the heat tolerance assay system
Tsubuku Co., Ltd., Kurume, Japan
Artificial rice paddy
Tsubuku Co., Ltd., Kurume, Japan
Width 1.33 m × depth 1.33 m × height 0.62 m, water capacity 510 L
Tsubuku Co., Ltd., Kurume, Japan
Diameter 7 cm, height 30 cm, 6 holes in 3 cm steps at each of the two sides
Metal halide lamp
Iwasaki Electric Co., Ltd., Tokyo, Japan
Toshiba Carrier Corp., Kawasaki, Japan
Temperature control tank
Tsubuku Co., Ltd., Kurume, Japan
Width 0.7 m × depth 0.7 m × height 1.52 m, water capacity 100 L
Sanso Electric Co., Ltd., Himeji, Japan
MH, HW, HS and SM conceived the project, and designed experiments. MH, CM-K and RT collected data. MH, HW and SM analysed the data and wrote the manuscript, and all authors contributed to revisions. All authors read and approved the final manuscript.
We thank Drs. Atsushi Maruyama and Hiroshi Nakano for many valuable suggestions during this study. We also thank Dr. Tomio Terao and NARO Genebank for providing seeds, and Ms. Kiyomi Koga, Ms. Kumika Kakiuchi, Ms. Fumie Tsuru and Ms. Fujiko Komiya for their technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
This work was supported by a Grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Project for Climate Change, Rice-1102), and by JSPS KAKENHI Grant Numbers 15K14644 and 16H04870.
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