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Gibberellin biosynthesis and signal transduction is essential for internode elongation

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in deepwater rice

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Running title: GA in deepwater rice

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Madoka Ayano1, Takahiro Kani1, Mikiko Kojima2, Hitoshi Sakakibara2, Takuya Kitaoka1,

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Takeshi Kuroha1, Rosalyn B. Angeles-Shim1, Hidemi Kitano1, Keisuke Nagai1 and

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Motoyuki Ashikari1†.

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1

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Aichi 464-8601, Japan.

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Bioscience and Biotechnology Center, Nagoya University, Furoucho, Chikusa, Nagoya,

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2

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Yokohama, Kanagawa, 230-0045, JAPAN

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Bioscience and Biotechnology Center, Nagoya University, Furoucho, Chikusa, Nagoya,

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Aichi 464-8601, Japan

RIKEN Center for Sustainable Resource Science, 1-7-22, Suehiro-cho, Tsurumi-ku,

Corresponding author: Motoyuki Ashikari

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E-mail address: [email protected]

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12377

1 This article is protected by copyright. All rights reserved.

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ABSTRACT

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Under flooded conditions, the leaves and internodes of deepwater rice can elongate above

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the water surface to capture oxygen and prevent drowning. Our previous studies showed

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that three major quantitative trait loci (QTL) regulate deepwater-dependent internode

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elongation in deepwater rice. In this study, we investigated the age-dependent internode

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elongation in deepwater rice. We also investigated the relationship between

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deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by

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physiological and genetic approach using a QTL pyramiding line (NIL-1+3+12).

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Deepwater rice did not show internode elongation before the sixth leaf stage under

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deepwater condition. Additionally, deepwater-dependent internode elongation occurred on

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the sixth and seventh internode during the sixth leaf stage. These results indicate that

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deepwater rice could not start internode elongation until the sixth leaf stage. LC-qMS/MS

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analysis showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice.

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Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in

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deepwater rice. On the other hand, GA feeding mimicked internode elongation under

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ordinary growth conditions. However, mutations in GA biosynthesis and signal

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transduction genes blocked deepwater-dependent internode elongation. These data

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suggested

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deepwater-dependent internode elongation in deepwater rice.

that

GA

biosynthesis

and

signal

transduction

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Key words: Deepwater rice, Gibberellin.

2 This article is protected by copyright. All rights reserved.

are

essential

for

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INTRODUCTION

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Flooding occurs during the rainy seasons in South Asia and South East Asia. In general, the

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leaves and internodes of general cultivated rice do not elongate during the vegetative stage,

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and when subjected to deepwater conditions, the plant dies due to oxygen starvation (Fig.

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1a and b). Deepwater rice shows a similar gross morphology (i.e. leaves and internodes are

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not elongated) as the general rice cultivar under shallow water conditions. However, during

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flooding, the leaves and internodes of deepwater rice elongate to avoid oxygen deficiency

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under rising water levels (Fig. 1c and d). The elongated internodes keep the top leaves

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above the water surface and aerenchyma formation in the internode supplies oxygen to the

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rest of the plant that is underwater. Internodes can elongate by up to 20-25 cm in a period

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of days (Catling. 1992). This characteristic has allowed deepwater rice to adapt to

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flood-prone areas.

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Physiological and genetic analysis has shown that the phytohormones ethylene and

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gibberellin are involved in internode elongation in deepwater rice. Under deepwater

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condition, enhanced ethylene biosynthesis and low diffusion of ethylene in water result in

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ethylene accumulation in the plant body. This ethylene accumulation triggers internode

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elongation in deepwater rice (Sauter and Kende. 1992, Hattori et al. 2009).

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The internode elongation ability of deepwater rice has been characterized based on

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three parameters: (1) total elongated internode length (TIL), (2) number of elongated

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internodes (NEI) and (3) lowest elongated internode (LEI) (Inouye and Mogami. 1980,

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Nemoto et al. 2004, Hattori et al. 2007, Kawano et al. 2008). Among these, LEI is a key

3 This article is protected by copyright. All rights reserved.

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parameter of internode elongation initiation in deepwater rice (Inouye and Mogami. 1980),

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because LEI is based on the leaf stage that first shows internode elongation.

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In this report, we explored the developmental stage in which the internode of

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deepwater rice could first elongate. At present, it remains unknown whether internode

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elongation can occur at any leaf stage during the vegetative phase and whether elongation

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occurs in any of the internodes. In this report, we determined whether specific internodes in

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deepwater rice elongate under deepwater conditions. Previous studies revealed that three

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major QTLs regulate deepwater responses (Nemoto et al. 2004, Tang et al. 2005, Hattori et

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al. 2007, Kawano et al. 2008). In this study, we used a QTL pyramiding line (NIL-1+3+12)

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carrying C9285 (deepwater rice) genomic fragments possessing three major QTLs on

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chromosomes 1, 3 and 12 in the T65 (general cultivated rice) genetic background to

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examine internode elongation patterns and initiation under deepwater conditions.

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Gibberellins (GAs) are a family of diterpenoids, and more than 100 GAs have

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been identified (MacMillan. 2002). Among them, only a small number, such as GA1 and

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GA4, are thought to be bioactive in plants. Bioactive GA regulates plant growth and

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development including seed germination, stem elongation, leaf expansion, and flower and

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seed development (Yamaguchi. 2008). GA deficient and GA signal deficient mutants (due

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to the lack of a GA biosynthetic gene and a GA signal transduction gene) in rice show a

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dwarf phenotype (Ross et al. 1997, Ueguchi-Tanaka et al. 2000, Itoh et al. 2002a, Itoh et al.

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2002b, Sakamoto et al. 2004). Exogenous GA treatment induces internode elongation of

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deepwater rice (Raskin and Kende. 1983, Suge. 1985, Hattori et al. 2009), suggesting that

4 This article is protected by copyright. All rights reserved.

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GA plays an important role in internode elongation. However, the mechanism of internode

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elongation in deepwater rice remains poorly understood.

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We also investigated the physiological and genetic interactions between GA and

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internode elongation in deepwater rice. We treated NIL-1+3+12 with active GA3 and

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developed mutant pyramiding lines possessing mutations in GA biosynthesis or signal

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transduction genes to determine whether GA is essential for internode elongation during the

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deepwater response. Moreover, we measured bioactive GAs and precursor concentrations,

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and determined the expression levels of GA biosynthetic and signal transduction genes. In

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this report, we show the internode elongation pattern during different leaf stages under

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deepwater conditions, as well as the physiological and genetic relationship between

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internode elongation and GA in deepwater rice.

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MATERIALS AND METHODS

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Plant materials and growth conditions

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Deepwater rice C9285 (O. sativa L.) was kindly provided by the National Institute of

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Genetics in Japan (http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp), whereas T65 (O.

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sativa L. ssp. japonica) was a cultivar maintained at Nagoya University. The QTL

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pyramiding line 1+3+12 (NIL-1+3+12) was previously produced at Nagoya University

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(Hattori. 2008, Hattori et al. 2009). Plant materials were grown in the greenhouse, growth

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chamber or paddy field in Nagoya University. In all experiments, seeds were pre-germinated at 29C in water for 3 days, and then

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sown in soil mixture (Mikawa baido) in plastic pots. At the 3 to 8 leaf stage (LS), seedlings

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were prepared for each experiment (Fig. S1). In this study, complete submergence

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conditions were applied to simulate deepwater conditions for 6 h to 14 days (Fig. S1).

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Shallow water condition was simulated by water levels less than 2 cm under the plant base

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(ordinary growth conditions). Deepwater condition represented complete submergence,

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which means that the entire plant body including the leaf tips remained underwater.

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Submergence, GA and uniconazole treatments

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All submergence, GA and GA biosynthesis inhibitor (uniconazole (Uni); inhibitor of

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ent-Kaurene oxidase) treatments were replicated in at least three independent biological

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experiments. The period of deepwater treatment varied. The 12-24h of deepwater treatment

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was performed for gene expression analysis (Fig. 7a–b and Fig. S3); the 7-day treatment

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was carried out for gross morphology analysis (Fig. 1e and f), internode length analysis

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(Fig. 2) and genetic analysis based on mutant pyramiding lines (Fig. 4); and the 14-day

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treatment was performed for GA treatment analysis (Fig. 4). A time course of submergence

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was performed for GA content analysis (Fig. 6). The GA feeding experiments (GA3 in

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Fig.4) was carried out in the growth chamber by exogenous application of GA3 at 10-5 M on

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the plants for 14 days. Shallow water (SW) and deepwater (DW) treatments were carried

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out by placing the plants in SW or DW conditions for 14 days. For the DW+Uni treatment,

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plants were pre-treated with 10-6 M Uni for 3 days before submerging them for 14 days (Fig.

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4). Experiments using mutant pyramiding line (see below) were performed in a greenhouse

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at Togo field of the Nagoya University. Other experiments were performed in a greenhouse

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and growth chamber of Nagoya University.

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Genetic analysis

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d18-dy [Waito-C, genetic background is ssp. Japonica.(cultivar name is unknown)]

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contains a mutation in the rice GA biosynthesis gene, OsGA3ox2, which catalyzes GA9 to

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GA4 and GA20 to GA1, and shows a weak dwarf phenotype (Itoh et al. 2002a, Sakamoto

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et al. 2004). gid1-7 (genetic background is ssp. japonica cv Nipponbare) and gid1-8

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(genetic background is ssp. japonica cv Taichung 65 (T65)) contain mutations in the rice

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GA receptor GID1. gid1-7 and gid1-8 are weak alleles of gid1 mutants that produce

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flowers (Ueguchi-Tanaka et al. 2007b). slr1-d1 (genetic background is ssp. japonica cv

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Taichung 65 (T65)) is a semi-dominant dwarf mutant that has mutation in the DELLA

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domain of the SLR protein. A mutation in the DELLA domain results in inefficient

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GA-dependent degradation of the SLR1 protein due to a reduced interaction with GID1

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(Asano et al. 2009). slr1-d1 mutants of rice exhibit a reduction in the length of all

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internodes but are reported to be fertile. gid2-2 and gid2-5 (genetic background is ssp.

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japonica cv Taichung 65 (T65)) contain mutations in the GA signaling factor, GID2, which

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is the F-box protein (E3 ligase) that targets SLR protein (Sasaki et al. 2003,

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Ueguchi-Tanaka et al. 2007). We obtained the mutant pyramiding (MP) lines that has the 3

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deepwater QTLs and GA homozygous mutations from an F2 population derived from

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crosses between the rice GA mutants (GA biosynthesis mutant and GA signal transduction

7 This article is protected by copyright. All rights reserved.

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mutant) and NIL-1+3+12. The MP lines were genotyped to confirm the presence of QTLs 1,

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3, 12 using the molecular markers used by Hattori et al. (2008) (Fig. S2). No background

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selection was carried out since all the GA mutants have a japonica background (T65 or

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Nipponbare except for d18-dy). The GA-related mutants were selected from the F1 and F2

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generations based on the dwarf phenotype and by sequence analysis. The MP lines were

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submerged for 7 days at the 8 LS, after which plant height and total internode elongation

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length (TIL) were measured. TIL was scored as the total length of internodes longer than 5

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mm.

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Measurement of phytohormone contents

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To measure endogenous phytohormone contents, ~200 mg of all aerial tissues except the

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developed leaf blade were collected before and after submergence. Each sample was

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collected into a 2.0 ml Master-Tube hard (Qiagen), frozen and crushed with four iron beads

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in liquid nitrogen. The concentration of endogenous GA was measured using LC-qMS/MS

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(UPLC-Xevo TQ-S;Waters) at the Institute of Plant Productivity Systems Research Group

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RIKEN Center for Sustainable Resource Science. Each compound was measured as

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described previously (Kojima et al. 2009).

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RNA isolation and expression analysis

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For semi-qRT-PCR and qRT-PCR analysis, samples were collected from the 7 LS of whole

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plants. Frozen samples were crushed and collected into liquid nitrogen. Total RNA was

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extracted with RNA solution using the RNeasy Plant mini kit (Qiagen). Total cDNA were

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obtained using TOYOBO-reverse transcription Ace (TOYOBO). For semi-qRT-PCR

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analysis, gene expression levels were analyzed using the Gene Amp PCR system 9700

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(Applied Biosystems). The quantile normalization method was employed using OsActin1

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as an internal control. For quantitative real-time RT-PCR (qRT-PCR) analysis of the

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expression of the GA biosynthesis genes in T65 and C9285 at submergence was carried out

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as described previously (Chomczynski and Sacchi. 1987). For the analysis, gene-specific

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primer sets (Li et al. 2011) and Sso Advanced SYBR Green Suprermix (BIO-RAD) were

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used. The refined gene expression was analyzed by Step One Plus (Applied Biosystems).

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Sequencing of regions flanking the primers used in the study confirmed that the primers

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can anneal to both T65 and C9285 genomic and cDNA. The gene-specific primers used for

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amplification in semi-qRT-PCR and qRT-PCR are described in Table S2.

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RESULTS

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Gross morphology and internode elongation patterns in deepwater rice

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We first compared the gross morphology among three rice lines namely, the general

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cultivated rice Taichung 65 (T65), the QTL pyramiding line (NIL-1+3+12) and deepwater

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rice variety, C9285 under shallow water and deepwater conditions (Fig. 1e and f).

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Differences in the gross morphology of T65, NIL-1+3+12 and C9285 were not observed

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before and after submergence (Fig. 1e). However, there were clear differences in internode

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elongation among the three lines (Fig. 1f). Internode (enclosed open yellow rectangle in Fig.

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1e) elongation was not observed in T65 under shallow water and deepwater conditions. On

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the other hand, we observed significant internode elongation under deepwater condition in

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NIL-1+3+12 and C9285. In C9285, internode elongation with pith cavity formation was

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observed under shallow water conditions, but elongated internode length was enhanced

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significantly under deepwater conditions (Fig. 1f). Based on these results, we examined the

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developmental stage at which internodes of deepwater rice could elongate. To accomplish

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this, deepwater rice at various leaf stages was monitored for internode elongation under

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deepwater conditions and the specific internode that first elongated under deepwater was

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identified.

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To evaluate deepwater rice traits, it is important to measure the total internode

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elongation length (TIL), number of elongated internodes (NEI) and lowest elongated

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internode (LEI) (Vergara and Mazaredo. 1979, Inouye and Mogami. 1980, Nemoto et al.

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2004, Tang et al. 2005, Hattori et al. 2007, Kawano et al. 2008). However, these

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measurements do not quantify each internode elongation. Here, we measured each

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internode length at different leaf stages before and after submergence to evaluate the

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developmental point (starting time) of the deepwater response. T65, NIL-1+3+12 and

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C9285 at the 3- 8 LS were submerged for 7days (Fig. S1). We measured the length of all

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internodes under shallow water and deepwater condition. Using this approach, we

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determined when internode elongation was initiated (Fig. 2a). Internode elongation was not

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observed under any condition from the 3 to 8 LS in T65 (Fig. 2b). The C9285 internode did

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not elongate (over 5 mm in length) from the 3 to 5 LS under any conditions, but the sixth

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and seventh internode showed elongation under deepwater condition at 6 LS (Fig. 2b). At

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the 7 LS of C9285, we also observed slight internode elongation at the seventh internode

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under shallow water conditions (Fig. 2b). Under shallow water conditions for C9285, the

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seventh internode length was 2.5 cm at the 7 LS, and the seventh and eighth internode

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lengths were 7.9 cm and 2.1 cm, respectively, at the 8 LS. However, more significant

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internode elongation was observed under deepwater conditions in C9285. The seventh and

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eighth internode lengths were 18.9 cm and 17.6 cm at the 7 LS, and the seventh, eighth and

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ninth internode lengths were 15.6 cm, 52.0 cm and 8.2 cm at 8 LS under deepwater

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conditions in C9285, respectively. In NIL-1+3+12, the eighth internode elongated by 9.1

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cm at the 7 LS, and the eighth and ninth internode elongated by 23.1 cm and 9.4 cm at the 8

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LS under deepwater conditions, respectively. Internode elongation patterns of C9285 under

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shallow and deepwater conditions were summarized in Fig. 3a-c.

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C9285 showed internode elongation at the 6 LS under deepwater conditions (Fig.

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3c), indicating that internode elongation occurs at 6 LS in C9285 under deepwater

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conditions. Under shallow water conditions, C9285 internode elongated slightly from the 7

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LS (Fig. 3b). However, the elongated length and number of internodes were higher under

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deepwater condition than shallow water condition after the 6 LS. Under deepwater

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condition, C9285 elongated two internodes at the 6 LS (the sixth and seventh internode)

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and 7 LS (seventh and eighth internode), and three internodes at the 8 LS (seventh, eighth

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and ninth internode). The sixth internode of C9285 at the 7 LS and 8 LS did not elongate.

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Internode elongation under deepwater conditions showed a good correlation with LS.

11 This article is protected by copyright. All rights reserved.

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Deepwater rice could initiate elongation at the sixth internode of C9285 at the 6 LS, but not

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at later leaf stages. This shows that the sixth internode has the potential to elongate under

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deepwater condition at the 6 LS, but may lose its internode elongation ability in later leaf

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stages. Thus, plants may use younger and upper internodes to achieve rapid internode

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elongation during emergency conditions, such as rapid flooding.

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Physiological relationship between gibberellins and internode elongation Previously, Raskin and Kende (1984) reported that 10 µM GA3 treatment of stem sections

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of deepwater rice induced internode elongation (Raskin and Kende. 1984). Suge (1985)

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also reported that 0.1, 1.0, and 10.0 ppm GA3 treatment of stem sections of three deepwater

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rice varieties the method slightly modified Raskin and Kende (1984) induced internode

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elongation. Recently, exogenous ethylene for stem section induced not only stem

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elongation, but also genes of GA metabolism enzymes (Choi. 2007). This suggests that GA

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plays a key role in internode elongation in deepwater rice. However, these experiments

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were performed using stem sections, so we investigated the role of GA in whole deepwater

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rice plants.

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To explore the mechanism of GA in internode elongation in deepwater rice, GA

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and its biosynthetic inhibitor, uniconazole were applied to T65, NIL-1+3+12 and C9285 at

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the 8 LS (Fig. 4a-c). Gross morphologies under four different conditions (shallow water,

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deepwater, deepwater plus 10-6 M uniconazole pretreatment, and 10-5 M GA treatment

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under shallow water condition) were shown in Fig. 4a. Significant plant height induction by

12 This article is protected by copyright. All rights reserved.

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GA treatment was observed in C9285, but not in T65 and NIL-1+3+12 (Fig. 4b). Total

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internode elongation was induced under deepwater conditions in NIL-1+3+12 and C9285,

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and this induction was abolished by uniconazole (Fig. 4c). GA treatment also induced

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internode elongation in NIL-1+3+12 and C9285 in shallow water condition. These

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physiological experiments suggested that GA treatment could mimic internode elongation

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in NIL-1+3+12 and deepwater rice (Fig. 4c). Additionally, GA is important for internode

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elongation in deepwater rice. However, these results do not confirm that GA is essential for

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internode elongation in deepwater rice because uniconazole blocks both a GA biosynthetic

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enzyme (P450) and other similar P450 enzymes (Iwasaki and Shibaoka. 1991, Asami et al.

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2001, Todoroki et al. 2008). It remains possible that other chemicals or hormones induce

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internode elongation under deepwater conditions in deepwater rice. We next used a genetic

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approach to determine whether GA is essential for internode elongation of deepwater rice

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under deepwater conditions.

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Genetic relationship between GA and internode elongation

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GA-deficient and GA signal deficient rice mutants have been isolated and characterized

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(Sakamoto et al. 2004, Ueguchi-Tanaka et al. 2007, Asano et al. 2009). We explored

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whether GA is essential for internode elongation in deepwater rice by introduction of

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characterized GA mutant alleles into the NIL-1+3+12 genetic background. Rice GA

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mutants (biosynthetic mutant, d18-dy: loss-of-function mutation of GA3oxidase; signal

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transduction mutant, gid1-7: loss-of-function mutation of GA receptor; gid1-8:

13 This article is protected by copyright. All rights reserved.

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loss-of-function mutation of GA receptor (weak allele); slr1-d1: gain-of-function mutation

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in DELLA domain; gid2-2: loss-of-function mutation of F-box; gid2-5, loss-of-function

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mutation of F-box (weak allele) (Gomi and Matsuoka. 2003, Sasaki et al. 2003,

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Ueguchi-Tanaka et al. 2008, Itoh et al. 2002, Asano et al. 2009) were crossed with

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NIL-1+3+12 to obtain the F2 (Supplemental Fig. 2). Mutant pyramiding lines possessing

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both the homozygous form of the GA mutant allele and the three major QTLs controlling

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deepwater response in plants at the vegetative stage were selected by genotyping and

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morphological selection for the dwarf phenotype. We grew the MP lines to the 8 LS since

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NIL-1+3+12 could elongate internodes at this stage (Fig. 2b). After 7 days of submergence,

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we observed the gross morphology and measured the plant height and total internode length

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of the MP lines (Fig. 5a-c). T65, NIL-1+3+12, d18-dy, and all the MP lines (d18-dy/

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NIL-1+3+12, gid1-7/ NIL-1+3+12, gid1-8/ NIL-1+3+12, slr1-d1/ NIL-1+3+12, gid2-2/

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NIL-1+3+12 and gid2-5/ NIL-1+3+12) showed a slightly increased plant height under

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deepwater condition (Fig. 5b). Total internode elongation in NIL-1+3+12 was significantly

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induced under deepwater condition (Fig. 5c). However, internode elongation was not

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observed in gid1-7/ NIL-1+3+12, gid2-2/ NIL-1+3+12 and gid2-5/ NIL-1+3+12 lines

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under deepwater condition. Very slight but significant differences (P

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

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our...
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