Spikelet opening under controlled transpiration conditions in rice
Funa Ogawa, Ryo Ishikawa, Takashige Ishii
Graduate School of Agricultural Science, Kobe University (1-1 Rokkodai, Nada-ku, Kobe 657 – 8501, Japan)
Summary: In rice, spikelet opening is an important process for anthesis to produce seeds. Spikelet opening is greatly affected by temperature, humidity, light, and physical stress. To clarify the important factors for spikelet opening, fl owering panicles of Japonica cultivar Nipponbare were observed under four controlled transpiration conditions designated as submergence, shower, oil and high-humidity conditions. The submergence condition was designed to allow transpiration only from panicles. Other three conditions were designed to suppress transpiration from panicles by water, oil and high humidity. For each condition, four panicles with 15 spikelets were prepared, and fl owering ratios were recorded on the following afternoon. The examination was repeated for three days, and the averages were compared. Under the submergence condition, many fl owered spikelets were observed with the average value of 83.3%. On the other hand, a few or no spikelets fl owered in the panicles under the shower (17.8%) and oil conditions (0.6%). Almost half of the spikelets (46.1%) fl owered under the high-humidity condition. These average values were all signifi cantly lower than the controls (ca. 92%) without any treatments. These results indicate that transpiration from lemma and palea is necessary to have spikelet opening. In addition, spikelet opening time was observed using a wild accession of O. rufi pogon to examine whether transpiration from an awn (a tip organ of the lemma) is associated with fl owering time or not. Eight panicles of three wild plants were prepared, and awns of the spikelets were alternatively cut based on the primary branch order. The plants were put outside under the sun, and the exact opening time was recorded in minutes for all the spikelets fl owered on the following morning. As a result, average opening times for awned and awnless spikelets were not signifi cantly different to each other. Since opening time was not associated with transpiration from the awns, a precise switch for panicle opening may synchronize with transpiration from the surface of the lemma and/or palea.
Keywords: Rice (Oryza sativa), Oryza rufi pogon, spikelet opening, transpiration, awn
Introduction
In rice, a spikelet has a basic floral unit composed of six stamens, one pistil, two lodicules, one lemma and one palea (Yamaguchi and Hirano 2006). The lodicules are a pair of small and thick organs at the base of the pistil inside the lemma. At the fl owering time, the lemma is mechanistically pushed outside by swollen lodicules though water absorption (Yoshida 2012). Spikelet opening is greatly affected by temperature, humidity, light, and physical stress (Tsuboi 1961, Kobayasi et al. 2010). Especially, bad weather conditions disturb spikelet opening. It is rare to observe opened spikelets on rainy days even in the peak fl owering period. These suggest that suppressed transpiration by high humidity and rain may directly cause low water absorption of the lodicules.
The spikelet flowering time is varied among cultivated and wild rice, and many wild accessions have early-morning
flowering traits (Sheehy et al. 2007). Using segregating populations between a Japonica cultivar Nipponbare and a wild accession of Oryza rufi pogon, Thanh et al. (2010) detected QTLs for spikelet fl owering time. Hirabayashi et al. (2015) also found a major QTL for early-morning flowering from O. officinalis, and further generated its near-isogenic lines. Since these lines flowered about two hours earlier than the Japanese cultivars, they have potential to avoid heat-induced spikelet sterility under future hotter climates. Flowering is an important process to produce seeds for the next generation, however, we do not know what kind of factors are associated with the species specific fl owering time.
Cultivated rice (O. sativa L.) was domesticated from the Asian wild species, O. rufipogon (Oka 1988). Compared with cultivated rice, wild rice produces spikelets with long awns (bristle-like organs at the tip of the lemmas) on spreading panicles. In the early stage of domestication, seed awning together with closed panicle shape are thought to be desirable for seed gatherers to collect mature seeds (Ishii et al. 2013).
Acccepted: January 20, 2019
Corresponding author: Takashige Ishii (tishii@kobe-u.ac.jp)
Research Article
However, once non-seed-shattering plants are generated, awnless phenotype may gradually appear because long awns disturb efficient harvesting and processing activities (Ikemoto et al. 2017). As a result, most of modern cultivars do not have awns. Since vigorous transpiration is expected from the awn, this organ might be related to spikelet opening time.
In this study, flowering panicles of Japonica cultivar Nipponbare were observed under controlled transpiration conditions to clarify important factors for spikelet opening. In addition, spikelet opening time was observed using a wild accession of O. rufi pogon to examine whether transpiration from an awn is associated with fl owering time or not.
Materials and methods
Plant materialsA Japonica cultivar, O. sativa Nipponbare, and a wild accession of O. rufi pogon W630 were used in this study. Their plants were grown in 1/5000 a pots, and spikelet fl owering was observed under several conditions.
Examination of spikelet opening of Nipponbare under four conditions
Spikelets of Nipponbare were prepared to examine major environmental factors for spikelet opening. At the peak of flowering period, 15 spikelets were selected in each panicle. They were expected to fl ower on the following morning based on the observation of anther position in the glumes. Then, other spikelets were cut off from the panicles in the afternoon (around 4:00 PM). The panicles were kept in the four environmental conditions, and the flowered spikelets were counted on the following afternoon (around 3:00 PM). For each condition, four panicles from one or two plants were examined on three days (Aug. 9 – 11, 2017). In each day, different plants were prepared for the examination.
The following four conditions were set to observe spikelet opening (Fig. 1).
- Submergence condition (Fig. 1A)
In the glasshouse, the whole plant except panicles was submerged in a plastic box (W 52.5 cm, D 37.5 cm, H 63.5 cm) fi lled with water, and the upper part of the box was tightly covered with a transparent plastic bag. Four panicles on a single plant were put outside from small holes of the plastic bag (Fig. 2A). The plant was subjected to this treatment around 5:00 PM. In this condition, the plants could not have transpiration except from the panicles.
- Shower condition (Fig. 1B)
A single plant was put under the water nozzle (LFX09-2560, Kohnan Co.) in the glasshouse (Fig. 2B). From the nozzle, water mist (about 500 ml per min) was produced, and the whole plant was wet as in the rain. This treatment was set at 8:30 AM (about two hours before start fl owering) on the same day of examination.
- Oil condition (Fig. 1C)
Only in this condition, four panicles were prepared from two plants (two panicles each). At around 5:00 PM, cooking oil (Canola oil, Ajinomoto Co.) was sprayed on the surface of the spikelets (Fig. 2C), and the plants were kept in the glasshouse. In this condition, transpiration was completely suppressed from lemma and palea.
- High-humidity condition (Fig. 1D)
The plants were put in a plastic box (W 52.5 cm, D 37.5 cm, H 63.5 cm) with some water at the bottom. Wet paper towels were hung inside, and the box was covered with a transparent plastic bag (Fig. 2D). In order to prevent rapid increase in temperature, the box was kept outside under the shade. The plant was subjected to this treatment around 5:00 PM. In the night time, relative humidity (RH) was 100% in the box. But it slightly decreased with an increase in temperature after sunrise. In the daytime, high humidity (more
Mistshower Oilcoated Water Wet towel Water
$
%
&
'
Fig. 1 Schematic representation of four controlled transpiration conditions to observe spikelet opening. (A) Submergence. (B) Shower. (C) Oil. (D) High humidity. Treated panicles under examination are shown in black.
than 77% RH) condition was maintained in the box tightly covered with a plastic bag. In this treatment, the whole plant was under high-humidity condition.
Besides the above plants under the four conditions, control plants without any treatments were also prepared in the glasshouse and outside under the shade. In those conditions, temperature and humidity were recorded every 5 min using thermo recorders (TR-72nw, T&D Co.).
Survey of spikelet opening time of wild rice
Several wild plants of O. rufipogon W630 in 1/5000 a pots were transferred from the glasshouse to outside just before heading period. Of these, eight panicles of three plants were selected to survey spikelet opening time. These panicles were at the same stage of flowering, i.e., at third day after starting fl owering. In each panicle, awns of spikelets were alternatively cut based on the primary branch order (Fig. 3). This awn treatment was performed at 3:00 PM, and the plants were continuously kept outside under the sun. On the following morning, the exact opening time was recorded in minutes for all the spikelets fl owered.
Statistical analysis
For spikelet opening experiment, average numbers of opened spikelets among four panicles were counted, and they were converted to flowering ratios by dividing by the total number. Using three-day averages, t-test was carried out to compare control values to those under four conditions at 5% signifi cant level.
For wild rice experiment, average opening times between awned and awnless spikelets were compared by t-test at 5% signifi cant level.
$
%
&
'
Fig. 2 Plants under the four conditions.
(A) Submergence: Four treated panicles were put outside from small hales of the plastic bag. (B) Shower: The plant was set in the mist shower. (C) Oil: Cooking oil was sprayed on the surface of the spikelets. (D) High humidity: The plant was kept in the plastic box tightly covered with the plastic bag.
Fig. 3 Wild panicles used to record spikelet opening time. Awns of spikelets were alternatively cut based on the primary branch order.
Results and discussion
Weather condition for spikelet opening of Nipponbare on the examination days
Spikelet opening of Nipponbare was examined under the four controlled transpiration conditions from Aug. 9 to 11, 2017. On these days, the weather was fi ne, and maximum and minimum temperatures were varied in the range of 36.4 - 38.6 ℃ and 24.0 - 26.8 ℃ , respectively. The average numbers of opened spikelets ranged from 13.5 to 14.0 and from 13.3 to 14.3 for the control plants in the glasshouse and outside, respectively (Table 1). This indicates that the weather condition was the same for spikelet opening on three days.
Spikelet opening of Nipponbare under the four controlled conditions
In the afternoon on the examination days, fl owered spikelets were counted in the panicles under the different conditions.
Table 1 shows numbers of flowered spikelets and flowering ratios. The average flowering ratios of three days were 92.2% and 92.8% for the control plants in the glasshouse and outside, respectively. Under the submergence condition, many fl owered spikelets were observed with the average value of 83.3%. On the other hand, a few or no spikelets fl owered in the panicles under the shower (17.8%) and oil conditions (0.6%). Almost half of the spikelets (46.1%) fl owered under the high-humidity condition. These average values were all significantly lower than the controls by t-test (p<0.05 for the submergence and high-humidity conditions, p<0.001 for the shower and oil conditions). The above results suggest that spikelets hardly flower if transpiration is completely suppressed from lemma and palea. Under the shower condition, whole plants were wet but some spikelets fl owered. On the other hand, most spikelets fl owered even though transpiration is completely suppressed except from the panicles under the submergence condition, confi rming that transpiration from lemma and palea is important for spikelet opening. Under high-humidity condition, half of the spikelets could flower, probably because of the partial suppression of transpiration from lemma and palea.
Survey of spikelet opening time of O. rufi pogon W630 with/ without awn treatment
Spikelet opening time of O. rufipogon W630 was surveyed under the sun on Aug. 27, 2017. In eight panicles of three plants, a total of 25 awned and 28 awnless spikelets were fl owered. Box plots of their spikelet opening time were shown in Fig. 4. The spikelet opening peak was around 8:30 to 9:00 AM, as previously observed on sunny days in August (Thanh et al. 2010). The average opening times for awned and awnless spikelets were 8:40 AM ± 19 min and 8:37 AM ± 31 min, respectively. They were not significantly different to each other (p=0.646), indicating that spikelet opening time was not associated with the awns.
In rice, spikelets fl ower only once in the morning. Therefore, spikelet opening is a very important process for anthesis to produce seeds. Probably, a precise switch for swelling lodicules may synchronize with transpiration from lemma and palea. An awn is a tip organ of the lemma, and is known to have transpiration through rows of stomata on surface (Li et al. 2010), indicating that awnless spikelets may have lower total transpiration than awned ones. If further suppression experiments of transpiration are performed for lemma and palea, we may know which organ is more responsible for spikelet opening.
References
Hirabayashi, H., K. Sasaki, T. Kambe, R. B. Gannaban, M. A.
$ZQHG$ZQOHVV
Q $YH 6' PLQ PLQFig. 4 Box plots of spikelet opening time for the awned and awnless spikelets. The central rectangles span the fi rst quartile to the third quartile, with the line indicating the median. The whiskers above and below the box show the locations of the minimum and maximum values.
1) Cont.: Control. Sub.: Submergence. Humid.: High humidity.
2) * and ***: Signifi cant at 5% and 0.1%, respectively, compared to the control.
Table. 1 Numbers of spikelets fl owered and fl owering ratios under four controlled transpiration conditions on three days.
'DWH
1R &RQW 6XE 6KRZHU 2LO &RQW +XPLG
$XJ $YH $XJ $YH $XJ $YH 2YHUDOO $YH 6' WWHVW 2XWVLGH *ODVVKRXVH S \
Miras, M. S. Mendioro, E. V. Simon, P. D. Lumanglas, D. Fujita, Y. Takemoto-Kuno, Y. Takeuchi, R. Kaji, M. Kondo, N. Kobayashi, T. Ogawa, I. Ando, K. S. Jagadish and T. Ishimaru (2015) qEMF3, a novel QTL for the early-morning fl owering trait from wild rice, Oryza offi cinalis, to mitigate heat stress damage at fl owering in rice, O. sativa. J. Exp. Bot. 66: 1227-1236.
Ikemoto, M., M. Otsuka, P. T. Thanh, P. D. T. Phan, R. Ishikawa and T. Ishii (2017) Gene interaction at seed-awning loci in the genetic background of wild rice. Genes Genet. Syst. 92: 21-26.
Ishii, T., K. Numaguchi, K. Miura, K. Yoshida, P. T. Thanh, T. M. Htun, M. Yamasaki, N. Komeda, T. Matsumoto, R. Terauchi, R. Ishikawa and M. Ashikari (2013) OsLG1 regulates a closed panicle trait in domesticated rice. Nat. Genet. 45: 462-465. Kobayasi, K., T. Marsui, M. Yoshimoto and T. Hasegawa (2010)
Effects of temperature, solar radiation, and vapor-pressure defi cit on fl ower opening time in rice. Plant Prod. Sci. 13: 21-28.
Li, X. F., D. Bin and H. A. Wang (2010) Awn anatomy of
common wheat (Triticum aestivum L.) and its relatives. Caryologia 63: 391-397.
Oka, H. I. (1988) The ancestors of cultivate rice. In Origin of cultivated rice , Japan Scientific Societies Press, Tokyo/ Elsevier, Amsterdam. 15-24.
Sheehy, J. E., A. E. Mabilangan, M. J. A. Dionora and P. P. Pablico (2007) Time of day of fl owering in wild species of the genus Oryza. Int. Rice Res. Notes 32: 12-13.
Thanh, P. T., P. D. T. Phan, N. Mori, R. Ishikawa and T. Ishii (2010). QTL analysis for flowering time using backcross population between Oryza sativa Nipponbare and O. rufi pogon. Genes Genet. Syst. 85: 273-279.
Tsuboi, Y. (1961) Ecological studies on rice plants with regard to damages caused by wind. Bull. Nat. Inst. Agr. Sci. A 8: 1-156. (in Japanese with English abstract)
Yamaguchi, T. and H. Y. Hirano (2006) Function and diversification of MADS-box genes in rice. Sci. World J. 6: 1923-1932.
Yoshida, H. (2012) Is the lodicule a petal: Molecular evidence? Plant Sci. 184: 121-128.
蒸散を制御した条件下におけるイネの小穂開花について
小川風和・石川 亮・石井尊生
神戸大学大学院農学研究科（〒 657-8501 神戸市灘区六甲台町 1-1） 要旨：イネにおいて小穂開花は種子生産に直結する受粉のための重要な過程のひとつである．小穂開花には，温度，湿度，光お よび物理的なストレスが大きく影響している．そこで，小穂開花に必要な要因を明らかにするために，まず日本型イネ品種の日 本晴を用いて，4 つの蒸散を制御した条件下（水没，シャワー，油，高湿度条件と表記）で小穂の開花調査を行なった．水没条 件では，植物体の穂のみからの蒸散が可能な状態を設定した．その他の 3 条件では，水，油，高湿度により穂からの蒸散を制御 するように設定した．それぞれの条件につき，開花直前の 15 小穂を残した 4 穂を処理し，翌日の午後に開花率を調査した．そ して，同じ調査を 3 日間繰り返し，それぞれの処理区の開花率の平均値を比較した．水没条件下では多くの小穂が開花し，平均 83.3% の開花率であった．一方，シャワーおよび油処理の条件下ではほとんど開花が見られなかった（シャワー：17.8%，油：0.6%）． また，高湿度条件下ではほぼ半数（46.1%）の小穂の開花が観察された．これらの平均開花率はそれぞれ無処理のコントロール 区（約 92%）のものと比較すると有意に低い値となった．これらの結果は，内穎と外穎からの蒸散は小穂の開花に必要であるこ とを示唆するものであった．次に，野生イネを用いて，外穎の先端に位置する芒からの蒸散が開花時間に関与しているか否かを 調査した．3 個体から 8 穂を準備し，1 次枝梗ごとに交互に小穂の芒を切除した．植物体を野外の自然条件下に配置し，翌朝開 花した全ての小穂の正確な開花開始時刻を分刻みで記録した．その結果，芒の有無によって開花開始時間に有意な差がないこと がわかった．芒からの蒸散は直接開花開始時間には関係しなかったが，小穂の開花の正確なスイッチは内穎または外穎の表面か らの蒸散に同調しているものと思われた．キーワード：イネ（Oryza sativa） ，Oryza rufi pogon，小穂開花，蒸散，芒
作物研究 64 号（2019） 連絡責任者：石井尊生（tishii@kobe-u.ac.jp）
