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. 2014 Jun 24;37(10):2313–2324. doi: 10.1111/pce.12377

Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

Madoka Ayano 1, Takahiro Kani 1, Mikiko Kojima 2, Hitoshi Sakakibara 2, Takuya Kitaoka 1, Takeshi Kuroha 1, Rosalyn B Angeles-Shim 1, Hidemi Kitano 1, Keisuke Nagai 1, Motoyuki Ashikari 1
PMCID: PMC4282320  PMID: 24891164

Abstract

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice.

Deepwater rice obtained the ability for rapid internode elongation to avoid drowning and adapt to flooded condition. How does it regulate internode elongation? Using both physiological and genetic approach, this paper shows that the plant hormone, gibberellin (GA) regulates internode elongation.

Keywords: gibberellin

Introduction

Flooding occurs during the rainy seasons in South Asia and South East Asia. In general, the leaves and internodes of general cultivated rice do not elongate during the vegetative stage, and when subjected to deepwater (DW) conditions, the plant dies due to oxygen starvation (Fig. 1a,b). Deepwater rice shows a similar gross morphology (i.e. leaves and internodes are not elongated) as the general rice cultivar under shallow water (SW) conditions. However, during flooding, the leaves and internodes of deepwater rice elongate to avoid oxygen deficiency under rising water levels (Fig. 1c,d). The elongated internodes keep the top leaves above the water surface and aerenchyma formation in the internode supplies oxygen to the rest of the plant that is underwater. Internodes can elongate by up to 20–25 cm in a period of days (Catling 1992). This characteristic has allowed deepwater rice to adapt to flood-prone areas.

Figure 1.

Figure 1

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Diagram and gross morphology of general cultivated rice, NIL-1 + 3 + 12 and deepwater rice before and after submergence. Diagram of the general cultivated rice T65 (a) and deepwater rice C9285(c) under shallow water (SW) and deepwater (DW) conditions. Arrowhead represents water level. (b) Node and internode components in general cultivated rice. Internode elongation was not induced in general cultivated rice under DW conditions. (d) Node and internode components in deepwater rice. Internode elongation induced in deepwater rice under DW conditions. (e) Gross morphology showing increased plant height before (SW) and after 7 d of DW treatment in T65, NIL-1 + 3 + 12 and C9285. Graphical genotypes are shown below the plant. Green solid rectangle indicates the T65 fragment and red solid rectangle indicates C9285 fragments. Yellow open rectangle shows the base of the internode. (f) Magnified base of internode shown in (e). Longitudinal sections showing only the basal part of the plant composed of the nodes and internodes. Pith cavities (*) are present in NIL-1 + 3 + 12 under DW and C9285 under SW and DW conditions. Scale bars: 5 mm in (f), 5 cm in (e).

Physiological and genetic analysis has shown that the phytohormones ethylene and gibberellin (GA) are involved in internode elongation in deepwater rice. Under DW condition, enhanced ethylene biosynthesis and low diffusion of ethylene in water result in ethylene accumulation in the plant body. This ethylene accumulation triggers internode elongation in deepwater rice (Sauter & Kende 1992; Hattori et al. 2009).

The internode elongation ability of deepwater rice has been characterized based upon three parameters: (1) total elongated internode length (TIL); (2) number of elongated internodes (NEI); and (3) lowest elongated internode (LEI) (Inouye & Mogami 1980; Nemoto et al. 2004; Hattori et al. 2007; Kawano et al. 2008). Among these, LEI is a key parameter of internode elongation initiation in deepwater rice (Inouye & Mogami 1980) because LEI is based upon the leaf stage that first shows internode elongation.

In this report, we explored the developmental stage in which the internode of deepwater rice could first elongate. At present, it remains unknown whether internode elongation can occur at any leaf stage during the vegetative phase and whether elongation occurs in any of the internodes. In this report, we determined whether specific internodes in deepwater rice elongate under DW conditions. Previous studies revealed that three major QTLs regulate DW responses (Nemoto et al. 2004; Tang et al. 2005; Hattori et al. 2007; Kawano et al. 2008). In this study, we used a QTL pyramiding line (NIL-1 + 3 + 12) carrying C9285 (deepwater rice) genomic fragments possessing three major QTLs on chromosomes 1, 3 and 12 in the T65 (general cultivated rice) genetic background to examine internode elongation patterns and initiation under DW conditions.

GAs are a family of diterpenoids, and more than 100 GAs have been identified (MacMillan 2002). Among them, only a small number, such as GA1 and GA4, are thought to be bioactive in plants. Bioactive GA regulates plant growth and development, including seed germination, stem elongation, leaf expansion, and flower and seed development (Yamaguchi 2008). GA-deficient and GA signal-deficient mutants (due to the lack of a GA biosynthetic gene and a GA signal transduction gene) in rice show a dwarf phenotype (Ross et al. 1997; Ueguchi-Tanaka et al. 2000; Itoh et al. 2002a,b,; Sakamoto et al. 2004). Exogenous GA treatment induces internode elongation of deepwater rice (Raskin & Kende 1983; Suge 1985; Hattori et al. 2009), suggesting that GA plays an important role in internode elongation. However, the mechanism of internode elongation in deepwater rice remains poorly understood.

We also investigated the physiological and genetic interactions between GA and internode elongation in deepwater rice. We treated NIL-1 + 3 + 12 with active GA3 and developed mutant pyramiding (MP) lines possessing mutations in GA biosynthesis or signal transduction genes to determine whether GA is essential for internode elongation during the DW response. Moreover, we measured bioactive GAs and precursor concentrations and determined the expression levels of GA biosynthetic and signal transduction genes. In this report, we show the internode elongation pattern during different leaf stages under DW conditions, as well as the physiological and genetic relationship between internode elongation and GA in deepwater rice.

Materials and Methods

Plant materials and growth conditions

Deepwater rice C9285 (Oryza sativa L.) was kindly provided by the National Institute of Genetics in Japan (http://www.shigen.nig.ac.jp/rice/oryzabase/), whereas T65 (O. sativa L. ssp. japonica) was a cultivar maintained at Nagoya University. The QTL pyramiding line 1 + 3 + 12 (NIL-1 + 3 + 12) was previously produced at Nagoya University (Hattori et al. 2008, 2009). Plant materials were grown in the greenhouse, growth chamber or paddy field in Nagoya University.

In all experiments, seeds were pre-germinated at 29 °C in water for 3 d, and then sown in soil mixture (Mikawa baido) in plastic pots. At the 3–8 leaf stage (LS), seedlings were prepared for each experiment (Supporting Information Fig. S1). In this study, complete submergence conditions were applied to simulate DW conditions for 6 h to 14 d (Supporting Information Fig. S1). SW condition was simulated by water levels less than 2 cm under the plant base (ordinary growth conditions). DW condition represented complete submergence, which means that the entire plant body including the leaf tips remained underwater.

Submergence, GA and uniconazole treatments

All submergence, GA and GA biosynthesis inhibitor [uniconazole (Uni); inhibitor of ent-Kaurene oxidase] treatments were replicated in at least three independent biological experiments. The period of DW treatment varied. The 12–24 h of DW treatment was performed for gene expression analysis (Fig. 7a,b and Supporting Information Fig. S3); the 7 d treatment was carried out for gross morphology analysis (Fig. 1e,f), internode length analysis (Fig. 2) and genetic analysis based upon MP lines (Fig. 4); and the 14 d treatment was performed for GA treatment analysis (Fig. 4). A time course of submergence was performed for GA content analysis (Fig. 6). The GA feeding experiments (GA3 in Fig. 4) was carried out in the growth chamber by exogenous application of GA3 at 10−5 m on the plants for 14 d. SW and DW treatments were carried out by placing the plants in SW or DW conditions for 14 d. For the DW + Uni treatment, plants were pre-treated with 10−6 m Uni for 3 d before submerging them for 14 d (Fig. 4). Experiments using MP line (see below) were performed in a greenhouse at Togo field of the Nagoya University. Other experiments were performed in a greenhouse and growth chamber of Nagoya University.

Figure 7.

Figure 7

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Semi-qRT-PCR and qRT-PCR analysis of GA biosynthesis gene expression analysis. (a) mRNA was obtained from the 7 LS of T65 and C9285 after 0, 12 and 24 h of submergence. PCR was performed for 30 and 35 cycles for GA biosynthesis and signal transduction genes and also 30 and 35 cycles for OsActin1. (b) qRT-PCR was performed using the SsoAdvanced SYBR Green Supermix with Step One Plus PCR system. Representation of the relative expression levels of GA20ox2 gene was normalized to the expression level of ubiquitin. The relative value of OsGA20ox2 expression in T65 at 0 h was 1. Vertical bars indicate SD of the mean of four replicates.

Figure 2.

Figure 2

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Leaf age-dependent internode elongation during submergence in T65, NIL-1 + 3 + 12 and C9285. (a) Diagram represents the longitudinal section of C9285 to show the internode position. (b) Deepwater response of T65 (green rectangle), NIL-1 + 3 + 12 (blue rectangle) and C9285 (red rectangle) was represented based upon comparison of the internode length during the three- to eight-leaf stage (3–8 LS) of the plant under shallow water (SW) and 7 d of deepwater (DW) treatment. X-axis indicates the position of the internode counted from the base to top, as shown in (a). Vertical bars indicate SD of the mean (n = 5–10) of three independent experiments.

Figure 4.

Figure 4

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Gibberellin (GA) feeding and elongation analysis in T65, NIL-1 + 3 + 12 and C9285. (a) Gross morphology of T65, NIL-1 + 3 + 12 and C9285 after treatment in 14 d shallow water (SW) and deepwater (DW), 3 d uniconazole (Uni) and 14 d DW (DW + Uni) treatment and 14 d 10−5 m of GA3 treatment (GA). (b, c) Plant height (b) and total internode length (c) in T65, NIL-1 + 3 + 12 and C9285 under SW (green bar), DW (blue bar), DW + Uni (red bar) and GA treatment (yellow bar) (n = 10); 3 independent experiments. Vertical bars indicated SE. Scale bars are 1 m in (a). Asterisks (*) indicate significant differences at P < 0.05 (Student's t-test).

Figure 6.

Figure 6

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Measurement of endogenous hormone concentrations under deepwater treatment at 7 LS in T65, NIL-1 + 3 + 12 and C9285. (a) Diagram represents the late step of GA1 and GA4 biosynthesis pathway to show enzymes and parallel relationships. (b–g) Endogenous levels of gibberellin (GA1) (d) and its precursors GA53 (b) and GA20 (c), as well as GA4 (g) and its precursors GA12 (e) and GA9 (f), were analysed after 0, 12, 24 and 48 h of deepwater treatment in T65 (green closed circle), NIL-1 + 3 + 12 (blue closed triangle) and C9285 (red closed rectangle) using UPLC-MS/MS (n = 4–7). Vertical bars indicate SD.

Genetic analysis

d18-dy [Waito-C, genetic background is ssp. Japonica (cultivar name is unknown)] contains a mutation in the rice GA biosynthesis gene, OsGA3ox2, which catalyses GA9 to GA4 and GA20 to GA1, and shows a weak dwarf phenotype (Itoh et al. 2002a; Sakamoto et al. 2004). gid1-7 (genetic background is ssp. japonica cv Nipponbare) and gid1-8 [genetic background is ssp. japonica cv Taichung 65 (T65)] contain mutations in the rice GA receptor GID1. gid1-7 and gid1-8 are weak alleles of gid1 mutants that produce flowers (Ueguchi-Tanaka et al. 2007b). slr1-d1 [genetic background is ssp. japonica cv Taichung 65 (T65] is a semi-dominant dwarf mutant that has mutation in the DELLA domain of the SLR protein. A mutation in the DELLA domain results in inefficient GA-dependent degradation of the SLR1 protein due to a reduced interaction with GID1 (Asano et al. 2009). slr1-d1 mutants of rice exhibit a reduction in the length of all internodes but are reported to be fertile. gid2-2 and gid2-5 [genetic background is ssp. japonica cv Taichung 65 (T65)] contain mutations in the GA signalling factor, GID2, which is the F-box protein (E3 ligase) that targets SLR protein (Sasaki et al. 2003; Ueguchi-Tanaka et al. 2007a). We obtained the MP lines that have the 3 DW QTLs and GA homozygous mutations from an F2 population derived from crosses between the rice GA mutants (GA biosynthesis mutant and GA signal transduction mutant) and NIL-1 + 3 + 12. The MP lines were genotyped to confirm the presence of QTLs 1, 3, 12 using the molecular markers used by Hattori et al. (2008) (Supporting Information Fig. S2). No background selection was carried out since all the GA mutants have a japonica background (T65 or Nipponbare except for d18-dy). The GA-related mutants were selected from the F1 and F2 generations based upon the dwarf phenotype and by sequence analysis. The MP lines were submerged for 7 d at the 8 LS, after which plant height and TIL were measured. TIL was scored as the total length of internodes longer than 5 mm.

Measurement of phytohormone contents

To measure endogenous phytohormone contents, ∼200 mg of all aerial tissues, except the developed leaf blade, was collected before and after submergence. Each sample was collected into a 2.0 mL Master-Tube hard (Qiagen, Turnberry Lane, Valencia, CA, USA), frozen and crushed with four iron beads in liquid nitrogen. The concentration of endogenous GA was measured using UPLC-MS/MS (UPLC-Xevo TQ-S; Waters, Maple Street, Milford, MA, USA) at the Institute of Plant Productivity Systems Research Group RIKEN Center for Sustainable Resource Science. Each compound was measured as described previously (Kojima et al. 2009).

RNA isolation and expression analysis

For semi-quantitative real-time RT-PCR (semi-qRT-PCR) and qRT-PCR analyses, samples were collected from the 7 LS of the whole plants. Frozen samples were crushed and collected into liquid nitrogen. Total RNA was extracted with RNA solution using the RNeasy Plant mini kit (Qiagen). Total cDNA were obtained using TOYOBO-reverse transcription Ace (TOYOBO, Dojima Hama, Kita-ku, Osaka, Japan). For semi-qRT-PCR analysis, gene expression levels were analysed using the Gene Amp PCR system 9700 (Thermo Fisher Scientific Inc., Wyman Street, Waltham, MA, USA). The quantile normalization method was employed using OsActin1 as an internal control. For qRT-PCR, analysis of the expression of the GA biosynthesis genes in T65 and C9285 at submergence was carried out as described previously (Chomczynski & Sacchi 1987). For the analysis, gene-specific primer sets (Li et al. 2011) and SsoAdvanced SYBR Green Supermix (Thermo Fisher Scientific Inc., Wyman Street, Waltham, MA, USA) were used. The refined gene expression was analysed by Step One Plus (Applied Biosystems). Sequencing of regions flanking the primers used in the study confirmed that the primers can anneal to both T65 and C9285 genomic and cDNA. The gene-specific primers used for amplification in semi-qRT-PCR and qRT-PCR are described in Supporting Information Table S2.

Results

Gross morphology and internode elongation patterns in deepwater rice

We first compared the gross morphology among three rice lines, namely the general cultivated rice Taichung 65 (T65), the QTL pyramiding line (NIL-1 + 3 + 12) and deepwater rice variety, C9285 under SW and DW conditions (Fig. 1e,f). Differences in the gross morphology of T65, NIL-1 + 3 + 12 and C9285 were not observed before and after submergence (Fig. 1e). However, there were clear differences in internode elongation among the three lines (Fig. 1f). Internode (enclosed open yellow rectangle in Fig. 1e) elongation was not observed in T65 under SW and DW conditions. On the contrary, we observed significant internode elongation under DW condition in NIL-1 + 3 + 12 and C9285. In C9285, internode elongation with pith cavity formation was observed under SW conditions, but elongated internode length was enhanced significantly under DW conditions (Fig. 1f). Based upon these results, we examined the developmental stage at which internodes of deepwater rice could elongate. To accomplish this, deepwater rice at various leaf stages was monitored for internode elongation under DW conditions and the specific internode that first elongated under deepwater was identified.

To evaluate deepwater rice traits, it is important to measure the TIL, NEI and LEI (Vergara & Mazaredo 1979; Inouye & Mogami 1980; Nemoto et al. 2004; Tang et al. 2005; Hattori et al. 2007; Kawano et al. 2008). However, these measurements do not quantify each internode elongation. Here, we measured each internode length at different leaf stages before and after submergence to evaluate the developmental point (starting time) of the DW response. T65, NIL-1 + 3 + 12 and C9285 at the 3–8 LS were submerged for 7 d (Supporting Information Fig. S1). We measured the length of all internodes under SW and DW conditions. Using this approach, we determined when internode elongation was initiated (Fig. 2a). Internode elongation was not observed under any condition from the 3–8 LS in T65 (Fig. 2b). The C9285 internode did not elongate (over 5 mm in length) from the 3–5 LS under any conditions, but the sixth and seventh internode showed elongation under DW condition at 6 LS (Fig. 2b). At the 7 LS of C9285, we also observed slight internode elongation at the seventh internode under SW conditions (Fig. 2b). Under SW conditions for C9285, the seventh internode length was 2.5 cm at the 7 LS, and the seventh and eighth internode lengths were 7.9 and 2.1 cm, respectively, at the 8 LS. However, more significant internode elongation was observed under DW conditions in C9285. The seventh and eighth internode lengths were 18.9 and 17.6 cm at the 7 LS, and the seventh, eighth and ninth internode lengths were 15.6, 52.0 and 8.2 cm at the 8 LS under DW conditions in C9285, respectively. In NIL-1 + 3 + 12, the eighth internode elongated by 9.1 cm at the 7 LS, and the eighth and ninth internode elongated by 23.1 and 9.4 cm at the 8 LS under DW conditions, respectively. Internode elongation patterns of C9285 under SW and DW conditions were summarized in Fig. 3a–c.

Figure 3.

Figure 3

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Elongation pattern of internode and leaves in C9285 after submergence. (a–c) Diagram of the three- to eight-leaf stage (3–8 LS) of C9285 under shallow water (SW) and deepwater (DW) conditions, showing gross morphology of the plant (a) and anatomical structure of the node and internode (b, c). (a) Gross morphology in the 3–8 LS of C9285 grown under SW conditions. (b, c) Position of internodes and leaves under SW (b) and DW (c) conditions. Only one leaf (marked in green L3-L8) represents the leaf stage. LS; leaf stage. L; leaf.

C9285 showed internode elongation at the 6 LS under DW conditions (Fig. 3c), indicating that internode elongation occurs at 6 LS in C9285 under DW conditions. Under SW conditions, C9285 internode elongated slightly from the 7 LS (Fig. 3b). However, the elongated length and number of internodes were higher under DW condition than SW condition after the 6 LS. Under DW condition, C9285 elongated two internodes at the 6 LS (the sixth and seventh internode) and 7 LS (seventh and eighth internode), and three internodes at the 8 LS (seventh, eighth and ninth internodes). The sixth internode of C9285 at the 7 LS and 8 LS did not elongate. Internode elongation under DW conditions showed a good correlation with LS. Deepwater rice could initiate elongation at the sixth internode of C9285 at the 6 LS, but not at later leaf stages. This shows that the sixth internode has the potential to elongate under DW condition at the 6 LS, but may lose its internode elongation ability in later leaf stages. Thus, plants may use younger and upper internodes to achieve rapid internode elongation during emergency conditions, such as rapid flooding.

Physiological relationship between GAs and internode elongation

Previously, Raskin & Kende (1984) reported that 10 μm GA3 treatment of stem sections of deepwater rice induced internode elongation (Raskin & Kende 1984). Suge (1985) also reported that 0.1, 1.0 and 10.0 ppm GA3 treatment of stem sections of three deepwater rice varieties [the method slightly modified by Raskin & Kende (1984)] induced internode elongation. Recently, exogenous ethylene for stem section induced not only stem elongation but also genes of GA metabolism enzymes (Choi 2007). This suggests that GA plays a key role in internode elongation in deepwater rice. However, these experiments were performed using stem sections, so we investigated the role of GA in whole deepwater rice plants.

To explore the mechanism of GA in internode elongation in deepwater rice, GA and its biosynthetic inhibitor, uniconazole, were applied to T65, NIL-1 + 3 + 12 and C9285 at the 8 LS (Fig. 4a–c). Gross morphologies under four different conditions (SW, DW, deepwater plus 10−6 m uniconazole pretreatment and 10−5 m GA treatment under SW condition) were shown in Fig. 4a. Significant plant height induction by GA treatment was observed in C9285, but not in T65 and NIL-1 + 3 + 12 (Fig. 4b). Total internode elongation was induced under DW conditions in NIL-1 + 3 + 12 and C9285, and this induction was abolished by uniconazole (Fig. 4c). GA treatment also induced internode elongation in NIL-1 + 3 + 12 and C9285 in SW condition. These physiological experiments suggested that GA treatment could mimic internode elongation in NIL-1 + 3 + 12 and deepwater rice (Fig. 4c). Additionally, GA is important for internode elongation in deepwater rice. However, these results do not confirm that GA is essential for internode elongation in deepwater rice because uniconazole blocks both a GA biosynthetic enzyme (P450) and other similar P450 enzymes (Iwasaki & Shibaoka 1991; Asami et al. 2001; Todoroki et al. 2008). It remains possible that other chemicals or hormones induce internode elongation under DW conditions in deepwater rice. We next used a genetic approach to determine whether GA is essential for internode elongation of deepwater rice under DW conditions.

Genetic relationship between GA and internode elongation

GA-deficient and GA signal-deficient rice mutants have been isolated and characterized (Sakamoto et al. 2004; Ueguchi-Tanaka et al. 2007a; Asano et al. 2009). We explored whether GA is essential for internode elongation in deepwater rice by the introduction of characterized GA mutant alleles into the NIL-1 + 3 + 12 genetic background. Rice GA mutants [biosynthetic mutant, d18-dy: loss-of-function mutation of GA3oxidase; signal transduction mutant, gid1-7: loss-of-function mutation of GA receptor; gid1-8: loss-of-function mutation of GA receptor (weak allele); slr1-d1: gain-of-function mutation in DELLA domain; gid2-2: loss-of-function mutation of F-box; gid2-5, loss-of-function mutation of F-box (weak allele); Gomi & Matsuoka 2003, Sasaki et al. 2003, Ueguchi-Tanaka et al. 2008, Itoh et al. 2002a; Asano et al. 2009] were crossed with NIL-1 + 3 + 12 to obtain the F2 (Supporting Information Fig. S2). MP lines possessing both the homozygous form of the GA mutant allele and the three major QTLs controlling DW response in plants at the vegetative stage were selected by genotyping and morphological selection for the dwarf phenotype. We grew the MP lines to the 8 LS since NIL-1 + 3 + 12 could elongate internodes at this stage (Fig. 2b). After 7 d of submergence, we observed the gross morphology and measured the plant height and total internode length of the MP lines (Fig. 5a–c). T65, NIL-1 + 3 + 12, d18-dy, and all the MP lines (d18-dy/NIL-1 + 3 + 12, gid1-7/NIL-1 + 3 + 12, gid1-8/NIL-1 + 3 + 12, slr1-d1/NIL-1 + 3 + 12, gid2-2/NIL-1 + 3 + 12 and gid2-5/NIL-1 + 3 + 12) showed a slightly increased plant height under DW condition (Fig. 5b). Total internode elongation in NIL-1 + 3 + 12 was significantly induced under DW condition (Fig. 5c). However, internode elongation was not observed in gid1-7/NIL-1 + 3 + 12, gid2-2/NIL-1 + 3 + 12 and gid2-5/NIL-1 + 3 + 12 lines under DW condition. Very slight but significant differences (P < 0.01; Student's t-test) in internode elongation was observed in d18-dy/NIL-1 + 3 + 12, gid1-8/NIL-1 + 3 + 12 and slr1-d1/NIL-1 + 3 + 12 lines under DW conditions (Fig. 5c). These results suggest that mutation in the GA biosynthesis gene or GA signal transduction gene abolished the ability for internode elongation in NIL-1 + 3 + 12 (Fig. 5c). This also suggested that blocking GA biosynthesis or signal transduction inhibits internode elongation even when the plant contains the three major QTLs involved in DW response. We next explored the type and concentration of GA produced under DW conditions in deepwater rice.

Figure 5.

Figure 5

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Gross morphology and deepwater response of mutants and mutant pyramiding lines after 7 d of deepwater treatment. (a) Gross morphology of GA biosynthesis (d18-dy), GA signal transduction (gid1-7, gid1-8), DELLA (slr1-dy), and F-box (gid2-2, gid2-5) mutants and mutant pyramiding (MP) lines after 7 d of shallow water (SW) and deepwater (DW) treatments. (b, c) Plant height (b) and total internode length (c) after 7 d of SW and DW conditions were determined (n = 8–10). Vertical bars indicate SE. Asterisks represents significant differences; *P < 0.05 and **P < 0.01 (Student's t-test). ND in (c) means not detected. Scale bar is 1 m in (a).

GA production in deepwater rice

GA1 and GA4 are active GAs in plants (Yamaguchi 2008). A late step of the GA biosynthetic pathway is illustrated in Fig. 6a. GA20oxidase (GA20ox) catalyses three steps from GA53 to GA20 and GA12 to GA9. Additionally, GA3oxidase (GA3ox) catalyses the final step from GA20 to GA1 and GA9 to GA4 (Fig. 6a). Hirano et al. (2008a) reported that different active GAs control plant growth; GA1 (predominant bioactive molecule) controls vegetative organ growth, whereas GA4 (dominant bioactive molecule) controls the reproductive rice organs (Hirano et al. 2008a). Kojima et al. (2009) reported that endogenous GA4 accumulates at the flower and panicle branch and not at the internode. It has also been reported that accumulation of GA4 induces an uppermost internode elongation during the heading stage of general cultivated rice, but not in vegetative organs (Magome et al. 2013). Hattori et al. (2009) reported that endogenous GA1 accumulates at the base of the stem in deepwater rice C9285 after submergence. However, the type and concentration of GA that induced internode elongation in deepwater rice at submerged condition are still unknown.

To examine changes in the GA level and the types of GAs that accumulate after submergence in deepwater rice, we employed mass spectrometry (MS) analysis. Both GA1 and GA4 accumulated in the aerial parts except in the developed leaves of NIL-1 + 3 + 12 and C9285 at after submergence (Fig. 6d,g and Supporting Information Table S1). Minor GA1 and GA4 accumulation was observed in T65, but GA production in C9285 was higher than in T65 (Fig. 6d,g and Supporting Information Table S1). The maximum GA1 and GA4 levels in C9285 after 24 h of submergence were similar (7–8 pmol g−1 FW). Additionally, time-dependent accumulation patterns of GA1 and GA4 were similar in C9285 (Fig. 6d,g). The time-dependent production of GA20 and GA9 (precursors of GA1 and GA4) were also similar under DW conditions in C9285 (Fig. 6c,f). These results suggest that GA1 and GA4 production under DW conditions is C9285-specific, and both GA1 and GA4 play a role in internode elongation in deepwater rice.

The time-dependent production of GA1 and GA4 and their precursors GA20 and GA9 in NIL-1 + 3 + 12 were similar to C9285 (Fig. 6c,f,d,g). This suggests that internode elongation in NIL-1 + 3 + 12 and C9285 is due to production of active GA1 and GA4. It also suggests that the accumulated GA1 and GA4 is caused by at least one of the QTL loci (originating from C9285), since NIL-1 + 3 + 12 and C9285 shared three chromosome fragments around the three major QTLs controlling the DW response (Fig. 1e). Based upon these results, we explored why active GA1 and GA4 accumulated under DW conditions in deepwater rice. To accomplish this, we examined the expression of GA biosynthetic and signal transduction genes in deepwater rice.

The expression profile of GA biosynthetic genes in deepwater rice

Bioactive GAs are synthesized through sequential steps by specific enzymes from trans-geranylgeranyl diphosphate (GGDP) (Yamaguchi 2008). We examined the steady-state levels of transcripts from GA biosynthesis genes (OsCPS1, OsCPS2, OsKS2, OsKS5, OsKO2, OsKAO, Os13ox, OsGA20ox1, OsGA20ox2, OsGA20ox3, OsGA3ox1, OsGA3ox2) (Magome et al. 2013), GA metabolic genes (OsGA2ox1, OsGA2ox3, OsGA2ox6, EUI, EUIL4) (Zhu et al. 2006) and signal transduction genes (OsGID1, OsGID2, OsSPY, OsSEC, OsGAMYB) (Tsuji et al. 2006; Ueguchi-Tanaka et al. 2007a; Hirano et al. 2008b; Phanchaisri et al. 2012) in whole plants of T65 and C9285 before and after submergence (Fig. 7 and Supporting Information Fig. S3).

Comparison of most of GA-associated gene transcript levels revealed no significant differences in whole T65 and C9285 plants (Fig. 7a). However, differences in the transcript accumulation of the GA biosynthesis gene OsGA20ox2 were observed between T65 and C9285. The expression of OsGA20ox2 in C9285 was highly up-regulated and prolonged after submergence. However, the transcript accumulation of OsGA20ox2 in T65 is only slightly up-regulated (Fig. 7a). qRT-PCR analysis showed a higher transcript accumulation of OsGA20ox2 in C9285 compared with T65 in DW condition (Fig. 7b). OsGA20ox2 catalyses GA53 to GA20 and GA12 to GA9 (Fig. 6a). The amount of GA20 and GA9 increased after submergence in C9285 (Fig. 6c,f), which corresponded to the induction of GA20ox gene expression in C9285 (Fig. 7b). Expression of GA metabolic genes and signal transduction genes were also observed, although gene transcript accumulation in T65 and C9285 did not differ when subjected to SW and submerged conditions (Supporting Information Fig. S3). Internode elongation in C9285 under DW conditions may be caused by GA1 and GA4 accumulation after induction of the GA20ox gene in plants under deepwater.

Discussion

To characterize the internode elongation mechanism in deepwater rice, we determined the developmental stage at which internode elongation occurs in deepwater rice under DW conditions. Internode elongation under DW conditions was first detected at the sixth internode at 6 LS and not before that (Figs 3). Based upon this result, an anatomical phase change in internodes or GA biosynthesis (or GA-associated processes) may occur between the 5 and 6 LS in C9285. For example, intercalary meristem (IM) located in the nodes could be activated, which can induce cell division and subsequently produce new cells. IM activation, which induces cell division and expansion, is a driving force in internode elongation. We propose three hypotheses to describe the relationship between IM and internode elongation (Fig. 8a–d). For these results, the longitudinal section was anatomically illustrated (Fig. 8a).

Figure 8.

Figure 8

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Schematic diagram representing three hypotheses of the IM activation mechanism. (a) The image shows a longitudinal section of deepwater rice under shallow water (SW) and deepwater (DW) conditions. (b) Image represents hypothesis 1, where IM formation of deepwater rice started at the sixth internode in the 6 LS. (c) Image represents hypothesis 2, where IM formation initiated upon submergence. (d) Image represents hypothesis 3, where IM formed at every internode but remained dormant until submergence. The old internode disappeared in response to DW. Active (elongation ability) IM is indicated in pink. The dormant or non-elongated (absent elongation ability) IM is indicated in orange.

The first hypothesis is that C9285 does not contain IMs before the 6 LS, but produces IM at the 6 LS under natural conditions which can then be activated by DW condition (Fig. 8b). The second hypothesis is that C9285 does not contain IMs at the nodes under SW conditions, but submergence induces IM production at the 6 LS (Fig. 8c). The last hypothesis is that C9285 establishes IMs at every node during an early leaf stage, but the IM remains dormant until the 6 LS until such time that it is activated by submergence (Fig. 8d).

Ikeda et al. (2001) reported that the GA constitutive signal transduction mutant (slr1-1: Nipponbare background) induced internode elongation from the third node. This single recessive mutant contains a single base deletion in the SLR1 gene causing a loss of function of the gene (Ikeda et al. 2001). This report suggests that IM could be established at least in the third node in T65. So based upon the result, hypothesis 1, in which deepwater rice establishes IM at the 6LS, is unlikely since deepwater rice requires rapid internode elongation to avoid flooding. Additionally, if the deepwater rice initiates IM after submergence (hypothesis 2), there will be a delay before internode elongation, which would put the plant at a high risk of drowning. Thus, hypothesis 2 is also unlikely. In the context of emergency conditions, hypothesis 3, in which deepwater rice establishes the IM at every node in an early leaf stage but remains dormant until submergence, is the most likely scenario.

We next explored how IM becomes established and activated at the sixth internode in deepwater rice. Plants become hypoxic under DW conditions, and it is known that ethylene gas accumulates in plants underwater (Kende et al. 1998; Hattori et al. 2009). Hypoxia, accumulation of ethylene and GA, or changes in the role of GA during submergence in C9285 may establish IM or activate dormant IM at the sixth node. In this study, GA was shown to be essential for internode elongation in deepwater rice. We also showed that GA accumulated in response to induction of the GA20ox gene. GA accumulation during submergence in C9285 may also activate dormant IM at the sixth node. Cell division and elongation would then induce internode elongation. Based upon these results, hypothesis 3 seems most likely, although hypotheses 1 and 2 remain possible. We do not clearly understand why deepwater rice can initiate internode elongation at the 6 LS. Additionally, the genesis of IM in deepwater rice remains poorly understood. Thus, the identification of IM specific markers and the study of IM in deepwater rice are required.

In the previous study, the QTL on chromosome 3 is detected based upon the LEI (Nemoto et al. 2004; Hattori et al. 2008; Kawano et al. 2008). The QTL on chromosome 3 determines the leaf stage that can initiate internode elongation. At this time, the QTL that regulates IM at the 6 LS has not been identified, but the QTL on chromosome 3 may be involved in activating or repressing IM.

C9285 showed slight internode elongation even under Sw condition (Figs 3). Under SW condition, C9285 would still produce ethylene or GA, which is induced by physical touch or wind. IM may have a high sensitivity to ethylene or GA in C9285, and could respond to low concentrations of ethylene or GA to induce internode elongation. The majority of elongated internodes under DW conditions correlated with the leaf stage (Figs 3). The sixth internode was highly elongated at the 6 LS, and the seventh internode was highly elongated at the 7 LS. However, at later leaf stages, the sixth and seventh internode of deepwater rice can no longer elongate. We observed that deepwater rice uses (activates) younger IMs for rapid internode elongation. Additionally, the internode elongation ability moves up the internode according to leaf stage, and old nodes may lack meristematic activity. The use of younger IMs for rapid internode elongation may be important to survive DW conditions.

UPLC-MS/MS analysis revealed that GA1 and GA4 accumulate not only in the leaf but also in the internode under DW conditions in C9285, but not in T65 (Fig. 6). Our analysis revealed that both GAs (GA1 or GA4) accumulate in the aerial parts including the internode, establishing the role of GAs in internode elongation. In case of GA1 production, GA53 in T65 is higher than that in C9285 under DW condition. On the contrary, the GA20 amount in C9285 is higher than that in T65 in deepwater (Fig. 6b,c). The higher expression of GA20ox2 in C9285 compared with T65 under DW condition may account for the observed GA53 accumulation in T65 and GA20 accumulation in C9285 (Fig. 7). The higher expression of GA20ox2 was consistent with the elevation of its product, GA1. GA9, GA4 and GA1 were detected in C9285 by MS (Fig. 6) but not GA12, probably because that the amount of GA12 produced was too low for detection by UPLC-MS/MS. Both GA1 and GA4 biosynthesis pathway is regulated by 13oxidase activity (Fig. 6a). However, the expression of GA13ox is not different in C9285 before and after DW condition. This stable expression may lead to both GA1 and GA4 accumulation. The expression of the GA metabolite gene, GA2ox6, was reduced in C9285 at 12 h. In a previous report, OsGA2ox6 has been identified to contribute to the inhibition of the vegetative stage of the plant, with its basal expression in T65 being very low (Lo et al. 2008). This indicates that metabolism of bioactive GAs (GA1 and GA4) in C9285 was also inhibited, thereby inducing internode elongation. In this study, the expression of GA3ox1 and GA3ox2, which are important in the final steps of GA biosynthesis, did not change before and after submergence in C9285 (Fig. 7a). These data suggest that the up-regulation of the GA20ox gene is an important step in generating active GA1 and GA4 in C9285. An eightfold increase in active GA accumulation is sufficient to enhance internode elongation. The current results on the transcript levels of GA metabolism genes were based upon the semi-qRT-PCR analysis and therefore are still preliminary. In the future, mRNA-sequence analysis might be able to shed additional light on the regulation of key genes in GA metabolism. For example, recent report from van Veen et al. (2014) revealed that two species of the eudicot genus Rumex, which inhabit different elevations of the Rhine River floodplain, display opposite submergence response strategies similar to what is observed in rice. Petiole elongation is exhibited in Rumex palustris, which endures prolonged deep floods, whereas this elongation is inhibited in Rumex acetosa, which only occasionally experiences short-term flooding. Unlike the scenario in rice, the petiole elongation was not evidently regulated by Group VII Ethylene response factors as in rice. Rather, R. palustris induced a distinct gene transcript profile as compared with rice that intersects with shade avoidance mediated petiole elongation. Interestingly, strong induction of the EIN3 BINDING F-BOX (EBF) OMCL gene family during submergence was observed only in R. palustris. The putative ortholog of rice is likely involved in ethylene-induced growth stimulation by preventing negative regulation of GA synthesis by ethylene (Kim et al. 2012; van Veen et al. 2013, 2014). Therefore, ethylene-regulated elongation may have diverged between eudicots and monocots. In monocots, IM is common; therefore, elongation of aerial part by cell elongation in IM had been reported in Avena (Kaufman 1965; Kaufman et al. 1965), Cyperus (Fisher 1970), Eleocharis (Evans 1965, 1969) and Zea mays (Sharman 1942). GA-induced internode elongation was also reported in Avena (Kaufman 1965). Recently, we detected that Brachypodium (Brachypodium distachyon) had also possessed GA responses and internode elongation (data not shown). Thereby, GA regulated elongation mechanism is also diverged among glass plants we thought. Future mRNA-sequence analysis of IMs of deepwater rice and other grass species may help to clarify ethylene- and GA-regulated elongation mechanisms.

In Fig. 4c, internode length in NIL-1 + 3 + 12 and C9285 in the presence of 10−5 m GA3 increased under DW conditions. This may be explained by the fact that 10−5 M GA3 is at a higher concentration than GA1 and GA4 under DW conditions in NIL-1 + 3 + 12 and C9285. The internode length of C9285 is greater than that of NIL-1 + 3 + 12 under DW conditions (Fig. 2). NIL-1 + 3 + 12 carry the three major QTLs that regulate internode elongation of C9285 in a T65 genetic background. However, an additional QTL is required to regulate internode elongation of DW condition in C9285. Nagai et al. (2012) detected two minor QTLs associated with internode elongation in C9285 (Nagai et al. 2012). Thus, the identification of other QTLs involved in internode elongation in deepwater rice is required.

Enhancement of leaf length by GA was similar in T65, NIL-1 + 3 + 12 and C9285 (Fig. 4b). GA treatment induced internode elongation in NIL-1 + 3 + 12 and C9285, but not in T65 (Fig. 4c). Both leaf and internode in NIL-1 + 3 + 12 and C9285 responded to GA. Leaves in T65 responded to GA, but internodes in T65 did not. These results suggest that organ-specific GA sensitivity differed between leaf (leaf sheath) and internode in T65, or that GA transport to the leaf and internode may differ between T65 and C9285.

Internode elongation was induced in NIL-1 + 3 + 12 by GA, suggesting that the QTL encodes genes responsible for GA sensitivity or transport. These could be important differences between non-deepwater rice and deepwater rice. Cloning and functional analysis of the QTLs could address the differences in leaf expansion and internode elongation by GA in T65.

Acknowledgments

We thank Dr Nori Kurata at the National Institute of Genetics (Mishima, Japan) and the NBRP Project for providing deepwater rice (C9285). This study was supported by Grant-in-Aid 22119001 (to M.A.) from the Ministry of Education, Culture, Sports, and Science and by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation, QTL-4002).

Supporting Information

Figure S1. Diagrams of methods of cultivation and deepwater treatment and experiments. (a) Deepwater treatment. Details of plant growth conditions were described in the Materials and Methods section. The three- to eight-leaf stages (3–8 LS) of plants were treated under shallow (SW) and complete submergence (DW). Plant height was measured to determine internode and leaf elongation, which represent the length from the base of the aerial part of the plant to the leaf tip. Total internode length represents the sum of all internode lengths in each plant. For gene expression analysis, stem sections containing young internode, shoot apical meristem and leaf sheath were collected.

Figure S2. Crossing method for the mutant pyramiding line. Parental line of GA biosynthesis and signal transduction mutants and NIL-1 + 3 + 12 were shown in the upper graphical genotype. The yellow closed rectangle represents the T65 genes, the red closed rectangle represents QTLs from C9285 and the blue closed rectangle represents GA-related genes. The F1 generations were obtained by crossing (represented as X). Backcrossing was performed several times. The homozygote plants of the MP line were finally selected based upon genotyping using molecular markers. The mutant pyramiding (MP) line possessed the three QTLs controlling the deepwater response and the GA-related genes as homozygotes.

Figure S3. Semi-qRT-PCR analysis of GA metabolic and signal transduction gene expression. mRNA was obtained from the 7 LS of T65 and C9285 after 0, 12 and 24 h of submergence. PCR was performed for 30 and 35 amplification cycles for GA metabolic and signal transduction genes expression and compared with that of OsActin1 amplified for 30 and 35 cycles.

Table S1. Adjusted P-values for the comparative GA contents in T65 and C9285 using the Holm's method. Significant data (P-value under 0.05; Student's t-test) was obtained by Holm's method. The data show GA1 and GA4 contents between T65 versus C9285 at 0, 6, 12, 24 and 48 h points and T65 versus NIL1 + 3 + 12 at 0, 6, 12, 24 and 48 h points.

Table S2. Sequence of primers used for the GA-related gene expression analysis using semi-qRT-PCR and qRT-PCR. Specific primers were designed for semi-qRT-PCR. Biosynthesis and signal transduction of GA were analysed as described previously.

pce0037-2313-sd1.pdf (708.9KB, pdf)

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Associated Data

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Supplementary Materials

Figure S1. Diagrams of methods of cultivation and deepwater treatment and experiments. (a) Deepwater treatment. Details of plant growth conditions were described in the Materials and Methods section. The three- to eight-leaf stages (3–8 LS) of plants were treated under shallow (SW) and complete submergence (DW). Plant height was measured to determine internode and leaf elongation, which represent the length from the base of the aerial part of the plant to the leaf tip. Total internode length represents the sum of all internode lengths in each plant. For gene expression analysis, stem sections containing young internode, shoot apical meristem and leaf sheath were collected.

Figure S2. Crossing method for the mutant pyramiding line. Parental line of GA biosynthesis and signal transduction mutants and NIL-1 + 3 + 12 were shown in the upper graphical genotype. The yellow closed rectangle represents the T65 genes, the red closed rectangle represents QTLs from C9285 and the blue closed rectangle represents GA-related genes. The F1 generations were obtained by crossing (represented as X). Backcrossing was performed several times. The homozygote plants of the MP line were finally selected based upon genotyping using molecular markers. The mutant pyramiding (MP) line possessed the three QTLs controlling the deepwater response and the GA-related genes as homozygotes.

Figure S3. Semi-qRT-PCR analysis of GA metabolic and signal transduction gene expression. mRNA was obtained from the 7 LS of T65 and C9285 after 0, 12 and 24 h of submergence. PCR was performed for 30 and 35 amplification cycles for GA metabolic and signal transduction genes expression and compared with that of OsActin1 amplified for 30 and 35 cycles.

Table S1. Adjusted P-values for the comparative GA contents in T65 and C9285 using the Holm's method. Significant data (P-value under 0.05; Student's t-test) was obtained by Holm's method. The data show GA1 and GA4 contents between T65 versus C9285 at 0, 6, 12, 24 and 48 h points and T65 versus NIL1 + 3 + 12 at 0, 6, 12, 24 and 48 h points.

Table S2. Sequence of primers used for the GA-related gene expression analysis using semi-qRT-PCR and qRT-PCR. Specific primers were designed for semi-qRT-PCR. Biosynthesis and signal transduction of GA were analysed as described previously.

pce0037-2313-sd1.pdf (708.9KB, pdf)

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