time-course transcriptomics analysis reveals key responses ... · time-course transcriptomics...

Time-Course Transcriptomics Analysis Reveals KeyResponses of Submerged Deepwater Rice to Flooding1

Anzu Minami,a,2 Kenji Yano,a,3 Rico Gamuyao,a,4 Keisuke Nagai,a Takeshi Kuroha,a,5 Madoka Ayano,a

Masanari Nakamori,a Masaya Koike,a Yuma Kondo,a Yoko Niimi,a Keiko Kuwata,b Takamasa Suzuki,c,d,6

Tetsuya Higashiyama,c,d,e Yumiko Takebayashi,f Mikiko Kojima,f Hitoshi Sakakibara,f,g Atsushi Toyoda,h

Asao Fujiyama,h Nori Kurata,i Motoyuki Ashikari,i and Stefan Reuschera,2

aBioscience and Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, JapanbInstitute of Transformative Bio-Molecules, Nagoya University, Nagoya, Aichi 464-8602, JapancGraduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, JapandERATO Higashiyama Live-Holonics Project, Nagoya University, Nagoya, Aichi 464-8602, JapaneInstitute of Transformative Bio-Molecules, Nagoya University, Nagoya Aichi 464-8601, JapanfRIKEN Center for Sustainable Resource Science, Tsurumi-ku, Yokohama 230-0045, JapangGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, JapanhCenter for Information Biology, National Institute of Genetics, Mishima 411-8540, JapaniGenetic Strains Research Center, National Institute of Genetics, Mishima 411-8540, Japan

ORCID IDs: 0000-0002-0112-6428 (R.G.); 0000-0003-4207-297X (Y.T.); 0000-0001-5449-6492 (H.S.); 0000-0002-0728-7548 (A.T.);0000-0002-8143-9300 (A.F.); 0000-0003-0491-5227 (S.R.).

Water submergence is an environmental factor that limits plant growth and survival. Deepwater rice (Oryza sativa) adapts tosubmergence by rapidly elongating its internodes and thereby maintaining its leaves above the water surface. We performed acomparative RNA sequencing transcriptome analysis of the shoot base region, including basal nodes, internodes, and shootapices of seedlings at two developmental stages from two varieties with contrasting deepwater growth responses. Atranscriptomic comparison between deepwater rice cv C9285 and nondeepwater rice cv Taichung 65 revealed both similarand differential expression patterns between the two genotypes during submergence. The expression of genes related togibberellin biosynthesis, trehalose biosynthesis, anaerobic fermentation, cell wall modification, and transcription factors thatinclude ethylene-responsive factors was significantly different between the varieties. Interestingly, in both varieties, the jasmonicacid content at the shoot base decreased during submergence, while exogenous jasmonic acid inhibited submergence-inducedinternode elongation in cv C9285, suggesting that jasmonic acid plays a role in the submergence response of rice. Furthermore, atargeted de novo transcript assembly revealed transcripts that were specific to cv C9285, including submergence-induced bioticstress-related genes. Our multifaceted transcriptome approach using the rice shoot base region illustrates a differential responseto submergence between deepwater and nondeepwater rice. Jasmonic acid metabolism appears to participate in thesubmergence-mediated internode elongation response of deepwater rice.

Submergence stress is harmful to plants. In additionto causing oxygen- and CO2-deficient conditions byrestricting environmental gas exchange, submergencereduces the light available for photosynthesis, perturbscellular energy generation, and disrupts ionic balance(Bailey-Serres and Voesenek, 2008; Voesenek and Bailey-Serres, 2015). The mechanism of the submergence re-sponse has been well studied using rice (Oryza sativa),two diverging Rumex spp. (Rumex acetosa and Rumexpalustris), and Arabidopsis (Arabidopsis thaliana), andseveral global analyses using transcriptomic or metab-olomic approaches to examine submergence or hypoxia/anoxia stress have been reported (Lasanthi-Kudahettigeet al., 2007; Magneschi and Perata, 2009; Mustroph et al.,2009, 2010; Narsai et al., 2009, 2015; Lakshmanan et al.,2013; van Veen et al., 2013; Rivera-Contreras et al., 2016).

Rice is the most important staple crop in Asia, andwater availability is a crucial factor for rice cultivation.

In tropical southeast Asia, rice is produced in paddyfields with water-controlling irrigation systems usingrivers, lakes, ponds, and swamps. However, in someparts of south and southeast Asia, such as Bangladesh,India, Thailand, Vietnam, and Cambodia, the paddyfields are frequently submerged during the rainy sea-son. The general cultivated rice cannot survive in thesesubmergence-prone areas, but some cultivars, such asfloating or deepwater rice, can grow and survive insuch conditions even under several-months-long pe-riods of deep flooding.

The adaptation of plants to submergence stressinvolves two different opposing mechanisms, the quies-cence strategy (e.g. rice SUBMERGENCE1 [SUB1] varie-ties, Arabidopsis ecotypes, and R. acetosa) and the escapestrategy (e.g. deepwater/floating rice varieties andR. palustris) (Jackson, 2008; Bailey-Serres et al., 2012; Loretiet al., 2016). Submergence-tolerant rice such as cv Flood

Plant Physiology�, April 2018, Vol. 176, pp. 3081–3102, www.plantphysiol.org � 2018 American Society of Plant Biologists. All Rights Reserved. 3081 www.plantphysiol.orgon October 16, 2020 - Published by Downloaded from

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http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0002-0728-7548

http://orcid.org/0000-0002-0728-7548

http://orcid.org/0000-0002-0728-7548

http://orcid.org/0000-0002-0728-7548

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0003-0491-5227

http://orcid.org/0000-0003-0491-5227

http://orcid.org/0000-0003-0491-5227

http://orcid.org/0000-0003-0491-5227

http://orcid.org/0000-0003-0491-5227

http://orcid.org/0000-0002-0112-6428

http://orcid.org/0000-0003-4207-297X

http://orcid.org/0000-0001-5449-6492

http://orcid.org/0000-0002-0728-7548

http://orcid.org/0000-0002-8143-9300

http://orcid.org/0000-0003-0491-5227

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Resistant 13A survives flooded conditions for a fewweeks through the quiescence strategy, wherein theplants temporarily cease shoot elongation to conserveenergy and then resume growth when the water recedes.Quantitative trait locus (QTL) mapping revealed that theSUB1 locus regulates the quiescence strategy (Fukao et al.,2006; Xu et al., 2006). The SUB1 locus on chromosome9 contains a cluster of three group VII ethylene responsefactor (ERF) genes (SUB1A, SUB1B, and SUB1C), and thepresence of the SUB1A-1 allele restricts underwater shootgrowth and confers submergence tolerance. SUB1A-1suppresses ethylene production, leading to the suppres-sion of GA synthesis andGA responsivenessmediated bythe negative regulators ofGAsignaling, SLENDERRICE1(SLR1) and SLR-LIKE1 (SLRL1; Fukao et al., 2006; FukaoandBailey-Serres, 2008).Moreover, SUB1A-1 activates theexpression of various genes, including other ERF genesand genes encoding transcription factors, reactive oxygenspecies scavengers, and enzymes involved in brassinos-teroid synthesis and several metabolic pathways facili-tating survival during the quiescent period (Fukao et al.,2006, 2011; Jung et al., 2010; Schmitz et al., 2013; Tamangand Fukao, 2015).

Conversely, the escape strategy involves stem elonga-tion to keep the leaves above the water surface. This typ-ically involves metabolic activation and the mobilizationof energy reserves to drive elongation growth (Fig. 1A).Utilizing this strategy, deepwater rice rapidly elongates itsinternodes (;20–25 cm per day) and reaches a length of

several meters in deep water (Catling, 1992; Kende et al.,1998). The deepwater rice cv C9285 from Bangladesh be-longs to the japonica varietal group (Wang et al., 2013) andshows strong internode elongation in response to sub-mergence (Hattori et al., 2009). Our previous QTL map-ping showed that the submergence-induced elongation incv C9285 is caused by three major QTLs located on chro-mosomes 1, 3, and 12 and two minor QTLs on chromo-somes 2 and 4 (Hattori et al., 2009; Nagai et al., 2012). Themajor QTL on chromosome 12 contains the two ERFfamily genes named SNORKEL1 and SNORKEL2 (SK1/2),which are positive regulators of internode elongation.During submergence, the gaseous hormone ethylene ac-cumulates, triggering SK1/2 gene expression in cv C9285.Although the downstream factors directly regulated bySK1/2 are still unknown, it is clearly established that in-ternode elongation in deepwater rice requires active GAbiosynthesis (Ayano et al., 2014; Nagai et al., 2014).

Both the underwater quiescence strategy and the es-cape strategy involve ethylene signaling and regulationby strategy-specific group VII ERFs: SUB1A limits stemelongation underwater, whereas SK1/2 promotes thesubmergence-induced elongation. The molecular mecha-nisms of the SUB1A-mediated quiescence strategy arewell studied (Bailey-Serres andVoesenek, 2010; Voesenekand Bailey-Serres, 2015), and SUB1-related gene expres-sion profiles during submergence have been analyzed(Jung et al., 2010; Fukao et al., 2011). However, there islittle information about the responses of deepwater riceunder the escape strategy. In this study, we explored thetranscriptional responses to submergence of the deep-water rice cv C9285. We aimed to compare the tran-scriptional responses associated with phytohormonesignaling, regulation of gene expression, and energy me-tabolism following the submergence of two contrastingrice varieties (deepwater and nondeepwater rice) at dif-ferent leaf developmental stages to better understand themechanism involved in the escape strategy. Our findingsexpose temporal and genotype-specific responses tosubmergence and implicate a role for jasmonic acid (JA)catabolism in the pronounced submergence-induced in-ternode elongation of deepwater rice.

RESULTS

Transcriptome Profiles of Deepwater Rice andNondeepwater Rice Plants Exposed to Submergence

To investigate the gene expression dynamics ofdeepwater rice in response to submergence, we com-pared the transcriptome profiles between the deepwa-ter rice cv C9285 and the nondeepwater rice cvTaichung 65 (T65; Supplemental Fig. S1). The inter-nodes of cv C9285 plants can elongate in response tosubmergence once plants reach the six-leaf stage (6LS;Ayano et al., 2014). Hence, for the transcriptome anal-ysis, cv C9285 and T65 plants at the 6LS were used forexpression profiling. We also included submerged cvC9285 plants at the four-leaf stage (4LS), which cannot

1 This work was supported by JST Core Research for EvolutionalScience and Technology, a MEXT Grant-in-Aid for Scientific Researchon Innovative Areas (22119007 and 17H06473) and JICA-JST SA-TREPS, by JSPS Grand-in Aid for Young Scientists (B) Grant Number17K15136 to A.M., and by a JST ERATO Grant (JPMJER1004) to T.H.

2 Address correspondence to [email protected] [email protected].

3 Current address: Faculty of Agriculture, Tokyo University, 1-1-1Yayoi, Bunkyo, Tokyo 113-8657, Japan.

4 Current address: Department of Ophthalmology, Johns HopkinsUniversity School of Medicine, Baltimore, MD 21287.

5 Current address: Department of Developmental Biology andNeurosciences, Graduate School of Life Sciences, Tohoku University,Sendai 980-8578, Japan.

6 Current address: College of Bioscience and Biotechnology,Chubu University, Matsumoto-cho, Kasugai, Aichi 478-8501, Japan.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Stefan Reuscher ([email protected]).

A.M., M.As., and S.R. designed this work and wrote the article; A.M. prepared all plant materials, made cDNA libraries, and performedphysiological experiments; A.M., K.N., T.K., M.Ay., M.N, Ma.K., Y.K., and Y.N. isolated RNA; T.S. and T.H. performed transcriptomesequencing, and S.R. analyzed the data; A.M. and K.Y. performedadditional data analysis; A.T., A.F., and N.K. provided C9285 ge-nomic sequence data, and S.R performed C9285 genome-targetedde novo assembly; K.Y. performed promoter element enrichmentanalysis; K.K. carried out proteomics analysis; Y.T., Mi.K., and H.S.analyzed hormone concentrations; R.G. provided many critical sug-gestions and did final editing and proofreading of the article.

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respond to submergence by elongation. The plantswere completely submerged for 0, 1, 3, 6, 12, and 24 h,and we sampled the shoot base region of the stem, asthis region in cv C9285 plants elongates rapidly inresponse to submergence. Per sample, on average,8.04 3 106 single-end reads were generated, of whichan average of 6.83 3 106 reads could be mapped to thetranscripts annotated in the japonica cv Nipponbarereference genome (Kawahara et al., 2013). The averagerates of mapped reads for cv C9285 and T65 samples inthe cv Nipponbare reference genome were 84.8% and86.1%, respectively (Supplemental Table S1). Afterquality control and filtering, we detected 27,812expressed genes in at least one sample (SupplementalData Set S1).

For global comparison of the transcriptomes derivedfrom all 18 tissue samples, we performed a principalcomponent analysis (PCA). The first two principalcomponents (PC1 and PC2) accounted for 48% of thetotal variance (Fig. 1B). PC1 accounted for 26% of allvariance in the data and clearly separated the cv T65samples (T65 6LS) from the cv C9285 samples at twodifferent stages (C9285 4LS and C9285 6LS). Among theT65 6LS samples, only the sample from the 24-h sub-mergence treatment showed lower PC1 values closer tothe cv C9285 samples. PC2 (accounting for 22% of allvariance) separated the samples based on the durationof submergence treatment. That is, each genotypeshowed higher and lower PC2 values, reflecting thesamples submerged for shorter and longer periods,

Figure 1. Submergence responses of deep-water rice and nondeepwater rice. A, Deep-water rice can survive and escape fromsubmerged conditions by rapid elongation ofstems (internodes) and leaves, while non-deepwater rice cannot elongate internodesduring submergence. B, PCA of 18 RNA-Seqsamples from cv C9285 and T65 submerged atdifferent leaf stages and time points. A plot ofall transcriptome samples along the first twoprincipal components (PC1 and PC2) isshown. The percentage of variation explainedis indicated at each axis. Colors indicatesamples from different genotypes and leafstages. Each data point represents averageddata from three independent time series, andone individual plant was sampled for eachdata point.

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respectively. The results from the PCA analysis suggestthat there are different gene expression patterns be-tween the two rice genotypes dependent on and inde-pendent of submergence treatment. Furthermore, theexpression profiles become variable depending on theduration of submergence treatment but were less af-fected by the leaf developmental stage in cv C9285plants. In addition, a more targeted attempt at isolatinggenes specifically induced by submergence at the 4LS,but not at the 6LS, in cv C9285 plants found only36 such genes (Supplemental Results S1).

Submergence induced expression changes for a largenumber of genes in both genotypes (SupplementalTable S2; Supplemental Fig. S2, A and B). To groupgenes with similar expression profiles, we performedk-means clustering of all 27,812 expressed genes. Weempirically chose k = 40 for k-means clusters andmanually grouped the clusters into four major groups:(1) higher expression in cv C9285 plants (1,019 genes;Supplemental Data Set S2); (2) higher expression in cvT65 plants (1,205 genes; Supplemental Data Set S3); (3)induced expression in both cv C9285 and T65 plants

(2,683 genes); and (4) repressed expression in both cvC9285 and T65 plants (4,125 genes), excluding clustersthat contained genes with no discernible pattern of ex-pression (Fig. 2A). In addition, to achieve a better sep-aration of submergence-responsive genes, we nextperformed k-means clustering (k = 20) for each geno-type separately (Supplemental Data Sets S4–S7). In cvC9285 samples, five (B6, B9, B11, B12, and B16) and four(B7, B8, B15, and B17) clusters contained submergence-induced and -repressed genes, respectively (Fig. 2B). Atotal of 2,429 genes in B6, B12, and B16 clusters showedthe strongest expression around 12 h of submergence,and a total of 775 early-responsive genes were classifiedinto B9 and B11 clusters. In cv T65 samples, five clusters(C1, C3, C5, C13, and C17) contained a total of 4,563submergence-induced genes, and most of the genesshowed relatively slow response to submergence, ex-cept for the 374 early-responsive genes in the C5 andC17 clusters (Fig. 2C). In cluster C2, the expression of514 genes in cv T65 plants was repressed by submer-gence. We evaluated the biological functions of genesenriched in each group of clusters based on the

Figure 2. Clustering analysis of the transcriptomes of cv C9285 and T65 plants after submergence. Heat map representations ofexpression data from selected clusters in both cv C9285 and T65 at the 6LS (C9286 6LS and T65 6LS; A) and from clusters only incv C9285 6LS (B) or cv T65 6LS (C) plants are shown. The clusters in the figure derived from Supplemental Data Set S1 werearranged manually. Rows represent clusters of genes generated by k-means clustering. Columns represent samples from differenttime points after submergence. Colors represent the average expression profile for each cluster, with red showing high expressionand blue showing low expression. Before averagingwithin each cluster, the expression of each genewas normalized to its averageexpression across all samples and transformed to log2 values. Numbers on the left side of each heat map show the assigned clusteridentifiers, and the number of genes in each cluster is in parentheses. The total number of clusters was k = 40 in A and k = 20 in Band C. FC, Fold change.


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MAPMAN ontology (Thimm et al., 2004; http://mapman.gabipd.org/web/guest/mapman). The hier-archical enrichment analysis of MAPMAN bins ofgenes with higher expression in cv C9285 or T65 plants(P, 0.05) and the data of submergence-changed genesin cv C9285 or T65 plants (P , 0.05) are shown inSupplemental Tables S3 and S4, respectively (describedin Supplemental Results S1). Selected enriched MAP-MAN bins were then chosen for further analysis.

Different Expression Patterns of Genes Related to PlantHormone Biosynthesis during Submergence inBoth Genotypes

Kende et al. (1998) proposed a model describing therelationship among the three phytohormones ethylene,abscisic acid (ABA), and GA in regulating rapid inter-node elongation of deepwater rice. During submer-gence, the reduced diffusion of ethylene causes itsaccumulation, which then triggers the ethylene signal-ing cascade, leading to the reduction of ABA contentand the increase of GA content (Kende et al., 1998;Fukao and Bailey-Serres, 2008). To investigate the genesrelated to hormone biosynthesis that are regulatedduring submergence, we compared the expressionlevels of hormone biosynthetic genes between cv C9285and T65 plants.Ethylene is produced from its precursor, S-adenosyl-

Met (SAM), derived from the Yang cycle through twosteps (Yang and Hoffman, 1984; Wang et al., 2002). Thefirst step is catalyzed by ACC synthase (ACS) con-verting SAM into 1-aminocyclopropane-1-carboxylicacid (ACC), while the second step involves ACC oxi-dase (ACO), which converts the ACC to ethylene(Rudus et al., 2013; Fig. 3A). The reaction catalyzed byACS is the major rate-limiting step of ethylene biosyn-thesis, and ACO also probably becomes the rate-limiting enzyme at low-oxygen and high-ethyleneconditions (Yang and Hoffman, 1984; Kende, 1993).Rice has six putative ACS genes (OsACS1–OsACS6;Rzewuski and Sauter, 2008), but only OsACS6 expres-sion was detected in our data set, showing a similarexpression pattern during submergence in both geno-types. In contrast, all seven rice ACO transcripts(OsACO1–OsACO7; Rzewuski and Sauter, 2008) weredetected. In both genotypes, OsACO1, OsACO2,OsACO3, and OsACO7 showed moderate to high ex-pression levels, while OsACO4, OsACO5, and OsACO6were expressed only at very low levels. OsACO7 wasinduced markedly by submergence in both genotypes,showing slightly higher expression in cv C9285 than incv T65. The expression profiles of genes involved inethylene signalingweremostly similar in both cv C9285and T65 plants (Supplemental Fig. S3A), which coin-cides with the similarly increased levels of ethyleneduring submergence in both genotypes (Hattori et al.,2009).GA is the key hormone for submergence-induced

stem elongation, and particularly, the concentrations of

bioactive GA1 and GA4 increase in submerged cv C9285plants (Hattori et al., 2009). GA biosynthesis starts fromthe conversion of trans-geranyl-geranyl diphosphatefollowed by four enzymatic steps to generate GA12 (Fig.3B; Richards et al., 2001; Yamaguchi, 2008). In the laterbiosynthetic steps, the conversion of GA12 to GA53 iscatalyzed by GA13 oxidase (GA13ox; Magome et al.,2013), and both GA12 and GA53 are catalyzed throughtwo parallel pathways (the early-13-hydroxylation andnon-13-hydroxylation pathways) by GA20ox. GA20oxcatalyzes the conversion of GA12 and GA53 into the bi-oactive GA precursors GA9 and GA20, respectively. Fi-nally, GA3ox converts GA9 and GA20 into the bioactiveGA forms GA4 and GA1. The expression patterns ofgenes encoding the enzymes before the catalytic step ofGA13ox were not prominently different between cvC9285 and T65 plants. GA20ox is the key enzyme ofbioactive GA synthesis, and rice has four putativeGA20ox genes (OsGA20ox1–OsGA20ox4; Sakamotoet al., 2004). The transcripts of three OsGA20ox geneswere detected in our data set, and only one of theOsGA20ox genes, OsGA20ox2, was strongly expressed1 h after submergence (greater than 15-fold increase) incv C9285 (Fig. 3B). On the other hand, in cv T65, theexpression level of OsGA20ox2was considerably lower(1.5 cpm in cv T65 versus 72 cpm in cv C9285 1 h aftersubmergence). OsGA20ox1 and OsGA20ox4 wereexpressed at low levels (less than 2.5 cpm) in bothgenotypes during submergence. Among the twoGA3oxgenes in rice, only OsGA3ox2 expression was detectedin both genotypes, showing a slight increase aftersubmergence. GA is inactivated by GA2ox andCYP714D1/EUI1 enzymes (Sakamoto et al., 2004; Zhuet al., 2006). Among the 11 putative OsGA2ox genes inrice, the expression of eight OsGA2ox genes wasdetected, and the expression patterns were mostlysimilar between the two genotypes. In our data, theexpression of CYP714D1/EUI1, which inactivates GA12,GA9, and GA4, could not be detected. We also investi-gated the expression profiles of GA signaling-relatedgenes during submergence and found that SLRL1 wasexpressed after submergence in both genotypes, al-though SLR1 was not induced by submergence(Supplemental Fig. S3B). However, the results remainelusive, because GA homeostasis interacts tightly withGAmetabolism and GA signaling pathways, which arecontrolled by feedback and feed-forward regulationsand various environmental factors (Sun, 2011; Heddenand Thomas, 2012).

ABA is a negative regulator of stem elongation duringsubmergence, corresponding to the decrease of ABAcontent in both submerged genotypes (Hoffmann-Benning and Kende, 1992; Hattori et al., 2009). In higherplants, the ABA precursor b-carotene is synthesized fromisopentenyl pyrophosphate through the plastidial meth-ylerythritol phosphate pathway (Endo et al., 2014).b-Carotene is catalyzed by several enzymes and eventu-ally converted intoABA (Fig. 3C). NCED is a rate-limitingenzyme in ABA biosynthesis, and in rice, NCED is pu-tatively encoded by five genes (OsNCED1–OsNCED5;




http://mapman.gabipd.org/web/guest/mapman

http://mapman.gabipd.org/web/guest/mapman






Zhu et al., 2009).OsNCED1,OsNCED2, andOsNCED5genes were detected in our data set, and the expressionof OsNCED2 was reduced in both genotypes 1 h aftersubmergence. Additionally, in the rice genome, thereare three genes that encode the ABA-inactivating en-zyme, ABA 89-hydroxylase (ABA8ox). In our data, theOsABA8ox1 and OsABA8ox2 genes in both genotypesshowed transiently increased expression after 1 h ofsubmergence. The genes involved in ABA signalingwere expressed similarly in both cv C9285 and T65plants (Supplemental Fig. S3C).

Hoffmann-Benning and Kende (1992) showed thatABA negatively regulates GA-mediated stem elongation

by experiments with excised stem sections of thedeepwater rice cv Habiganj Aman II. To test the effectof ABA on the GA activity in whole plants, cv C9285plants were grown from seeds for 18 d in shallowwater containing GA3, ABA, or a combination of bothGA3 and ABA. Treatment with GA3 increased thetotal plant height and internode length comparedwith untreated plants (Fig. 3D). While treatment withABA alone had no observable effect, treatment withcombined ABA and GA3 reduced the GA-inducedelongation of cv C9285 plants.

JAs control plant growth, development, and re-sponses to abiotic and biotic stresses. JAs are derived

Figure 3. Ethylene, GA, and ABAmetabolism during submergence. A to C, Schematic overviews of ethylene (A), GA (B), and ABA(C) metabolism alongside heat maps showing the expression of relevant genes. Each row represents one gene, columns representsamples from different time points after submergence, and colors represent gene expression levels as log2-transformed counts permillion (cpm). Lower levels of expression are represented in blue and higher expression in yellow. AAO3, ABA-aldehyde oxidase;ABA8ox, ABA-89 oxidase; CPS, copalyl-phosphate synthase; CYP714, cytochrome P714; GAXox, GAX oxidase; GGDP, geranyl-geranyl diphosphate; KAO, ent-kaurenoic acid oxidase; KO, ent-kaurene oxidase; KS, ent-kaurene synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; ZEP1, zeaxanthin epoxidase1. Asterisks indicates genes with significant differences (falsediscovery rate, 0.05) at more than four time points between cv C9285 and T65 samples, and, and. indicate the direction ofthe difference relative to cv C9285. D, Effects of ABA andGA3 on plant height and total internode length. The cv C9285 4LS plantswere grown in shallow water containing 10 mM GA3 and/or 10 mM ABA for 18 d. Bars represent averages of at least seven bio-logical replicates, and error bars show SE. Asterisks show significant differences (P , 0.05) between the indicated treatments ascalculated by Student’s t tests.


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from a-linolenic acid, which is released from gal-actolipids of chloroplast membranes by fatty aciddesaturase and phospholipase A1, including defective inanther dehiscence1 (Wasternack and Hause, 2013; Fig.4A). a-Linolenic acid is converted to OPC-8 through theaction of several enzymes, including lipoxygenase,allene oxide synthase, allene oxide cyclase, and 12-oxo-phytodienoic acid reductase3. After the three steps ofb-oxidation, OPC-8 is converted into jasmonoyl-CoAand then cleaved to (+)-7-iso-jasmonoyl by thioester-ase. Finally, (+)-7-iso-jasmonoyl-Ile, which is a ligandbinding a coreceptor of JA receptors, is catalyzed byjasmonate resistant1 in the cytosol. Through the activity

of the members of the CYP94 family, (+)-7-iso-jasmonoyl-Ile is inactivated through hydroxylation.The putative JA-inactivating CYP94C4 gene showedhigher expression during submergence compared withbefore submergence in both genotypes. In addition,endogenous JA levels decreased after 24 h of submer-gence in both genotypes (Fig. 4B). To examine the effectof JA on the submergence response, we submerged cvC9285 plants in water containing 50 mM methyl jasmo-nate andmeasured the total internode length after 3 d oftreatment (Fig. 4C). Surprisingly, treatment with JAremarkably inhibited the submergence-induced inter-node elongation.

Figure 4. JAmetabolism during submergence. A,Schematic overview of JA metabolism alongsideheat maps showing the expression of relevantgenes. Each row represents one gene, columnsrepresent samples from different time points aftersubmergence, and colors represent gene expres-sion levels as log2-transformed cpm. Lower levelsof expression are represented in blue and higherexpression in yellow. FDA, Fatty acid desaturase;DAD1, defective in anther dehiscence1; PLA,phospholipase A1; LOX, lipoxygenase; 13(S)-HPOT, 13(S)-hydroperoxyoctadecatrienoic acid;AOS, allene oxide synthase; 12,13-EOT, 12,13(S)-epoxy-octadecatrienoic acid; AOC, alleneoxide cyclase; OPDA, cis-(+)-12-oxo-phytodie-noic acid; OPR, 12-oxo-phytodienoic acidreductase; OPC-8, 3-oxo-2-(29-pentenyl)-cyclo-pentane-1-octanoic acid; OPCL1, acyl-activatingenzyme; ACX, acyl-CoA oxidase; MFP, multi-functional protein; KAT, 3-keto-acyl-CoA thio-lase; TS2, tasselseed2; JA-CoA, jasmonoyl-CoA;(+)-7-iso-JA, (+)-7-iso-jasmonoyl; JAR1, jasmo-nate resistant1; JA-lle, (+)-7-iso-jasmonoly-L-Ile;CYP94, cytochrome P94. Asterisks indicategenes with significant differences (false discoveryrate , 0.05) at more than four time points be-tween cv C9285 and T65 samples, and , and .indicate the direction of the difference relative tocv C9285. B, Endogenous JA levels in the shootbase regions of cv C9285 and T65 plants duringsubmergence for 24 h. Symbols represent aver-ages of at least five biological replicates, and er-ror bars represent SE. The asterisk shows asignificant difference (P , 0.05) between cvC9285 and T65 samples as calculated by Stu-dent’s t tests. FW, Fresh weight. C, JA inhibitsinternode elongation during submergence. Thecv C9285 6LS plants were submerged underwater containing 50 mM methyl jasmonate (Me-JA) for 3 d. Bars represent averages of at least12 biological replicates, and error bars show SE.The asterisk shows a significant difference (P ,0.05) between the indicated treatments as cal-culated by Student’s t tests.





Deepwater Treatment Has Genotype-Specific Effects onTrehalose Metabolism and Fermentation-Related Pathways

We further investigated the expression of genes inother relevant pathways to understand the metabolicadaptation of deepwater rice under submergence.Comparative analysis of metabolic pathways usingMAPMAN software showed that genes related to themetabolism of trehalose (Supplemental Fig. S4A) andfermentation (Supplemental Fig. S4B) were induced inresponse to 1 h of submergence in cv C9285 plants. Theexpression of genes in the NO3-generating pathway(Supplemental Fig. S4C) also was up-regulated aftersubmergence, but their absolute expression was at verylow levels.

Trehalose is a disaccharide that functions in carbonmetabolism and tolerance to biotic and abiotic stresses(Lunn et al., 2014). In plants, trehalose is synthesized ina two-step process from UDP-Glc and Glc-6-P by thesubsequent actions of trehalose-6-phosphate synthases(TPS) and trehalose-6-phosphate phosphatases (TPP),and the degradation of trehalose is catalyzed by tre-halase (TRE; Fig. 5A; Ponnu et al., 2011). In our data set,

we detected the expression of trehalose metabolism-related genes, including 13 TPS (out of 14), seven TPP(out of 13), and one TRE (Ge et al., 2008) genes. SomeTPS genes clearly responded to submergence in cvC9285 plants. Notably, three TPS genes (OsTPS12,OsTPS13, and OsTPS14) were strongly induced in re-sponse to submergence in cv C9285 plants but not in cvT65 plants (Fig. 5B). OsTPP1 and OsTPP11 showed el-evated expression in response to submergence in cvC9285 plants, while OsTPP2 expression was induced ata relatively low level in cv T65 after submergence (Fig.5C). In the case of TRE, the expression pattern wassimilar in both genotypes (Fig. 5D).

Under anaerobic conditions, oxygen is limited foraerobic respiration and plant cells implement anaerobicrespiration (glycolysis and fermentation) for energyproduction instead (Perata et al., 1998; Fukao andBailey-Serres, 2004; Bailey-Serres and Voesenek, 2008;Miro and Ismail, 2013). The glycolysis pathway gener-ates ATP, and the following fermentation steps produceNAD+ for the maintenance of glycolysis and somemetabolites. Fermentation converts the glycolysis-derived pyruvate into other metabolites such as Ala,

Figure 5. Expression of trehalose metabolism-related genes during submergence. A, Schematic overview of the trehalose met-abolic pathway. Glu-6P, Glc-6-P; UDPG, UDP-Glc. B to D, Expression of genes involved in trehalose metabolism during sub-mergence in cv C9285 6LS (black) and cv T65 6LS (orange) plants at the indicated time points after submergence. Data pointsshow expression levels as average cpm in three biological replicates, and error bars show SD. Black and orange asterisks indicatesignificant differences (P , 0.05) from 0 h at each time point for cv C9285 and T65 samples, respectively. Daggers indicatesignificant differences (false discovery rate , 0.05) between cv C9285 and T65 samples. P values were calculated using likeli-hood ratio tests and corrected for multiple testing by the Bonferroni-Holm method.


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lactate, ethanol, and acetate (Fig. 6A). Ala is synthe-sized from pyruvate and Glu by Ala aminotransferase(AlaAT), while lactate is formed by the reduction ofpyruvate via lactate dehydrogenase (LDH). Pyruvatedecarboxylase (PDC) converts pyruvate to acetalde-hyde. To detoxify acetaldehyde, alcohol dehydrogen-ase (ADH) or acetaldehyde dehydrogenase (ALDH)

subsequently catalyzes the conversion of acetaldehydeto ethanol or acetate, respectively. Interestingly, wefound that the two LDH genes (LDH-A and LDH-B)were strongly and specifically induced 1 h after sub-mergence in cv C9285 but not in cv T65 plants (Fig. 6B).Among other fermentation-related genes, OsAlaAT1,OsPDC1, OsPDC2, and OsPDC3, Os05g0469800,

Figure 6. Expression of fermentation-related genes during submergence. A, Schematic overview of three fermentation pathways.B, Expression of genes related to fermentation during submergence in cv C9285 6LS (black) and cv T65 6LS (orange) plants at theindicated time points after submergence. Data points show expression levels as average cpm in three biological replicates, anderror bars show SD. Graphs are arranged to reflect the different possibilities of anaerobic energy production. PEP, Phospho-enolpyruvate. Black and orange asterisks indicate significant differences (P, 0.05) from 0 h at each time point for cv C9285 andT65 samples, respectively. Daggers indicate significant differences (P, 0.05) between cv C9285 and T65 samples. P values werecalculated using likelihood ratio tests and corrected for multiple testing by the Bonferroni-Holm method.





OsADH1 and OsADH2, and OsALDH2a appeared toincrease after submergence in both genotypes, whereasgenes in the ALDH1 family seemed to show variableexpression patterns during submergence.

Submergence Induces Differential Expression Profiles ofGenes Related to Cell Wall Formation between cv C9285and T65 Plants

Under the nonsubmerged condition, genes related tocell wall formation and secondary metabolic pathwaysshowed higher expression in cv C9285 plants comparedwith cv T65 plants (Supplemental Fig. S5, A–D). Theplant cell wall provides mechanical strength, regulatesgrowth, serves as a barrier to diffusion, and protects theplant against pathogens. The cell wall-related tran-scripts with higher expression in cv C9285 plantsencoded cell wall modification proteins a- andb-expansins (EXPAs and EXPBs), xyloglucan endo-transglycosylases/hydrolases (XTHs), pectin esterases(PMEs), and structural cell wall proteins (fasciclin-likearabinogalactans [FLAs] and extensins; Fig. 7A). In cvT65 plants, almost all genes responded to submergence,and the expression level finally reached that of cv C9285within 24 h of submergence. In cv C9285 plants, severalgenes were strongly induced by submergence, espe-cially the genes EXPB6, EXPB7, OsXTH23, andOsXTH1, and two PMEs (Os01g0880300 andOs01g0312500), three FLAs (FLA7, FLA6, and FLA2),and two extensins (Os02g0138000 and Os01g0594300)remarkably responded to submergence in cv C9285plants.

Under nonsubmerged conditions, genes involved inflavonoid, anthocyanin, and phenylpropanoid metab-olism, as well as in the biosynthesis of lignin and simplephenols (e.g. laccases involved in lignin polymeriza-tion), were expressed higher in cv C9285 comparedwith cv T65 (Supplemental Fig. S5C). Lignin is a com-plex polymer of monolignols and strengthens the cellwall as a component of secondary cell walls. Thus, cellwall lignification probably functions in the suppressionof rapid growth in deepwater rice, resulting in a slowrate of elongation of its stems in nonsubmerged con-ditions (Sauter and Kende 1992). We investigated thetranscriptional responses of lignin biosynthesis-relatedgenes to submergence and focused on genes encodingconiferyl/sinapyl alcohol dehydrogenase (CAD/SAD),which catalyzes the final step of monolignol biosyn-thesis (Supplemental Fig. S6A). In cv C9285 plants,among the 11CAD/SAD genes (out of a total of 12 genesin the genome), OsCAD1, OsCAD2, OsCAD8B, andOsCAD8C were highly expressed in nonsubmergedconditions and, except for OsCAD1, showed decreasedexpression in response to submergence (Fig. 7B).OsCAD1 showed a similar expression level in bothgenotypes, while the other CAD genes in cv T65maintained lower expression levels during submer-gence. To determine whether the decrease in the ex-pression of CAD genes affects lignin biosynthesis in

elongating internodes of cv C9285 plants, we measuredthe lignin content of the cv C9285 stem, including (1)nodes from different positions, (2) nonelongated inter-nodes (an internode that already stopped elongating),and (3) newly elongated internodes (a newly elongatinginternode during submergence; Fig. 7C). Under thenonsubmerged condition, the basal node, non-elongated internode, and second and third nodesshowed similar lignin contents (Fig. 7D). The total lig-nin content in the basal and second nodes did notchange after 2 d of submergence (Fig. 7, D and E). Onthe other hand, the third node on the internode newlyelongated after 2 d of submergence exhibited a lowerlignin content (Fig. 7E). The total lignin content in thenonelongating internode was not affected by 2 d ofsubmergence, while the content of the newly elongatedinternodes was lower than that of the nonelongatedinternode. These results suggest that the suppression oflignin biosynthesis through the reduction of CADtranscripts contributes to the elongation of internodesin cv C9285 plants. In agreement with this, the lignincontent in the basal nodes of cv T65 plants did notchange during submergence (Supplemental Fig. S6B).

Expression of ERF Transcription Factor Genes in Responseto Submergence

The MAPMAN overrepresentation analysis revealedthat transcription factor genes from the AP2/EREBPfamily were enriched significantly among genesinduced by submergence in both genotypes(Supplemental Table S4, A and B). To investigate theresponses of all transcription factor genes to submer-gence, the genes in our data set were categorizedaccording to the Plant Transcription Factor Databaseversion 3.0 (http://planttfdb.cbi.pku.edu.cn/; Jin et al.,2014; Supplemental Data Set S8; Supplemental Fig. S7;Supplemental Results S1). Interestingly, those clusterscontained a total of 10 ERF genes (Supplemental Fig.S7). The ERFs belong to the AP2/EREBP superfamily,which is a plant-specific transcription factor familywiththree subfamilies: (1) AP2 family proteins containingtwo repeated AP2 DNA-binding domains; (2) ERFfamily proteins containing a single AP2 domain; and (3)RAV family proteins containing a B3 DNA-bindingdomain and a single AP2 domain (Riechmann andMeyerowitz, 1998; Sakuma et al., 2002; Nakano et al.,2006). Nakano et al. (2006) divided the rice ERF family(a total of 139 genes) into 15 subfamilies including28 subgroups. In our data set, we categorized the ERFgenes into these subgroups. Among all transcriptionfactors in our data set, 115 were found to be ERF genes,including 41, 10, and 64 genes with increased, de-creased, and unchanged expression during submer-gence, respectively (Supplemental Table S5). The ERFgenes from subgroups IIa, IIIc, VIIa, VIIb, IXa, Xa,Xb, Xc, and XI predominantly showed increased ex-pression in response to submergence in cv C9285plants, while those of subgroups IIIe and Va showed


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http://planttfdb.cbi.pku.edu.cn/








Figure 7. Effects of submergence on the expression of cell wall formation-related genes. A, Expression pattern of cell wall-relatedgenes during 24 h of submergence. The gene expression levels in cv C9285 6LS and cv T65 6LS samples are indicated by log2 foldchange (FC) at each time point (0, 1, 3, 12, and 24 h) relative to that of cv T65 samples before submergence. Geneswith amaximalcpm value of more than 15 are shown. AGP, Arabinogalactan protein. B, Changes in expression of 11 CAD/SAD genes during





decreased expression under submergence treatment.Supplemental Figure S8 shows expression profiles ofthe submergence-induced ERF genes. The SUB1 locusencodes three ERF transcription factor genes (SUB1A,SUB1B, and SUB1C; Xu et al., 2006); however, the ge-nome of cv C9285 contains only SUB1B and SUB1C butdoes not have SUB1A (Hattori et al., 2009). In cv C9285plants, SUB1B (OsERF#063) and SUB1C (OsERF#073)increased 3.2- and 2.7-fold 1 h after submergence, re-spectively (Supplemental Fig. S8A). The expression ofSK1 and SK2 genes, which are positive regulators ofinternode elongation in cv C9285 plants and absent incv T65 plants, also was increased remarkably 5.3- and3.2-fold by submergence, respectively (SupplementalFig. S8B).

GCC-Box-Like Promoter Elements Are Enriched in thePromoters of Genes Expressed Specifically in cvC9285 Plants

Using k-means clustering, we isolated 189 genes thatare expressed specifically in cv C9285 plants(Supplemental Data Set S9). Their putative promoterregions (1 kb upstream from the transcriptional startsite) were constructed by aligning reads from genomicDNA of cv C9285 plants to the japonica cv Nipponbarereference genome. The constructed promoter sequenceswere then analyzed for enriched DNA sequence motifsusing the MEME suite. We found two GCC-box likeelements (GCGGCGGCGG and CGCCGCCGCC)enriched in sequence among the analyzed promotersequences (Supplemental Fig. S9). The consensus DNA-bindingmotif (GCCGCC) is known to be recognized bythe ERF proteins (Hao et al., 1998; Fujimoto et al., 2000).

Identification and Analysis of Differentially ExpressedGenes Unique to cv C9285

Although about 85% of cv C9285 RNA sequencing(RNA-Seq) reads could be mapped to the cv Nippon-bare reference genome (Supplemental Table S1), thereis a possibility that cv C9285 has additional genesmissing in the reference genome, such as SK1 and SK2.Hence, we performed a targeted de novo assembly oftranscripts to detect cv C9285-specific transcripts(Supplemental Fig. S10; SupplementalMethods S1).Weused 47,978,704 paired-end reads from genomic DNAof cv C9285 (approximately 203 coverage) to assemble

C9285-specific genomic regions and quantified tran-scripts detected in those regions using RNA-Seq reads.We then restricted our analysis to those transcripts thatare expressed at high levels, are strongly induced bysubmergence, or have an interesting annotation. In to-tal, 86 transcribed loci on 33 genomic contigs werefound to be either putatively unique to cv C9285 orsufficiently different from the cv Nipponbare referenceso that mapping with standard parameters failed toproduce alignments (Supplemental Data Sets S10 andS12–S14).

Among those 86 loci, two encoded the full-length SK1and SK2 transcripts. The genomic contigs that con-tained the SK1 (contig_21; 5,776 bp) and SK2 (contig_8;12,025 bp) loci aligned almost perfectly with parts of thebacterial artificial chromosome clone that was used formap-based cloning of SK1 and SK2 (Hattori et al., 2009).When we quantified the expression of SK1 and SK2genes from those contigs, we found the typical earlyincrease of transcriptional activity 1 h after submer-gence with a subsequent decline (Supplemental Fig.S8B). This shows that our targeted assembly strategywas able to correctly identify unique cv C9285 tran-scripts, including their genomic sequence and theirexpression patterns. We found 11 novel transcripts thathad at least 2-fold higher expression levels after 1 h ofsubmergence (Fig. 8). Except for SK1 and SK2, most ofthose transcripts did not encode well-characterizedproteins. Instead, a sequence comparison usingBLASTP reported at least six pathogen- or disease-related proteins. The remaining three proteins wereannotated as domain of unknown function, retro-transposon related, and SAM-dependent methyltransferase.

DISCUSSION

The overall goal of this study was to understand thetranscriptional responses associated with the escapestrategy of the deepwater rice cv C9285 during sub-mergence. Cataloging thewhole transcriptome throughan RNA-Seq approach revealed differences in geneexpression between deepwater rice (cv C9285) andnondeepwater rice (cv T65) plants aswell as unique andcommon responses to submergence in the two geno-types. The results of our comprehensive transcriptomeanalysis contribute to the finding of key genes regu-lating the metabolic pathways during submergence inrice.

Figure 7. (Continued.)submergence for 24 h. Data points show expression levels as average cpm in three biological replicates, and error bars show SD.C, Sampled regions of internodes for lignin quantification. The cv C9285 plants at the seven-leaf stage (7LS) that have alreadyformed internodeswere submerged for 2 d. Images of stems before (0 d) and after (2 d) submergence treatment of cv C9285 plantsat the 7LS are shown. Red arrowheads indicate stem nodes. D and E, Lignin content in the nodes and internodes before (D) andafter (E) 2 d of submergence of cv C9285 plants at 7LS. Bars represent averages of at least six biological replicates, and error barsrepresent SE. Different letters indicate significant (P , 0.05) differences according to the Tukey-Kramer test.


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Transcripts Associated with Ethylene, GA, and ABAMetabolism Are Differentially Expressed in Responseto Submergence

Previous studies have demonstrated that the hor-mones ethylene, GA, and ABA modulate the internodeelongation response of submerged deepwater rice(Kende et al., 1998). Among the ethylene biosynthesisgenes, it is reported that OsACS1, OsACS5, andOsACO1were increased by submergence in deepwaterrice (Mekhedov and Kende, 1996; Zarembinski andTheologis, 1997; Van Der Straeten et al., 2001). On theother hand, in our data,OsACO7 transcript and proteinaccumulated during submergence in both genotypes(Fig. 3A; Supplemental Fig. S11A). Ethylene accumu-lation in submerged deepwater rice is attributed to thedecrease in ethylene diffusion and the enhancement ofethylene biosynthesis (Métraux and Kende, 1983). Ourprevious study showed that ethylene accumulation is acommon occurrence in both cv C9285 and T65 plants(Hattori et al., 2009). Our transcript analysis supportsthat OsACO7 is involved in ethylene accumulation inboth genotypes during submergence. OsACO7 has notbeen reported to participate in ethylene biosynthesisduring submergence, although most previous studiesused nondeepwater rice varieties.Under the submergence condition, the accumulated

ethylene enhances GA accumulation for the promotionof internode elongation in deepwater rice (Métraux andKende, 1983, 1984; Raskin and Kende, 1984; Kendeet al., 1998). Interestingly, unlike ethylene biosynthesis,the activation of GA biosynthesis occurs only in cvC9285 plants (Hattori et al., 2009). In our data,OsGA20ox2 (also known as SD1) expression levels werestrongly and specifically induced by submergence in cv

C9285 but not in cv T65 (Fig. 3B).GA20ox2 is a key locusfor GA production; thus, its increased expressionis most likely responsible for the higher GA1 andGA4 accumulation in submerged cv C9285 plants(Supplemental Fig. S12F; Hattori et al., 2009; Ayanoet al., 2014). In the case of the submergence-tolerant ricewith SUB1A, SUB1A suppresses GA signaling throughthe accumulation of GA signaling repressors, SLR1 andSLRL1, leading to the restricted elongation of stems andleaves under submergence (Fukao and Bailey-Serres,2008; Colebrook et al., 2014). Thus, the absence ofSUB1A in the deepwater rice cv C9285 may explain theaccumulation of bioactive GA levels promoting theelongation of stems and leaves during submergence. Ithas been reported that the GA-induced gene GrowthRegulating Factor1 (OsGRF1) and a GA stimulated tran-script (GAST)-like gene, OsGSR1, enhance stem elon-gation in rice (van der Knaap et al., 2000; Ben-Nissanet al., 2004; Choi et al., 2004; Wang et al., 2009). Inter-estingly, two GAST-like genes (Os05g0376800 andOsGSR1) were expressed higher in cv C9285 plantsduring submergence, although OsGRF1 was notinduced during submergence in our data set(Supplemental Fig. S11, B and C), suggesting that theGAST-like genes may be involved in GA-induced stemelongation during submergence in cv C9285 plants(Furukawa et al., 2006).

The content of ABA, which has the antagonistic effectto GA on internode elongation, decreased under sub-mergence and ethylene treatments (Hoffmann-Benningand Kende, 1992; Azuma et al.,1995; Benschop et al.,2006; Yang and Choi, 2006; Saika et al., 2007; Weiss andOri, 2007; Fukao and Bailey-Serres, 2008; Hattori et al.,2009; Chen et al., 2010). The decrease in ABA content isa common response to submergence in both cv T65 and

Figure 8. Submergence-induced transcripts from cv C9285-unique loci. Expression from selected loci that are presumably absentin the IRGSP-1.0 reference genome is shown. Each row represents one locus, columns represent samples from different timepoints after submergence, and colors represent gene expression levels as log2-transformed cpm normalized to the average ex-pression in all samples of different time points. Genes that have at least a 2-fold increase in expression after submergence in cvC9285 6LS plants are shown. Complete expression data from cv C9285-unique transcripts can be found in Supplemental Data SetS10, and sequence data are provided in Supplemental Data Sets S12 to S14. AA, Amino acids; FC, fold change.











C9285 plants (Hattori et al., 2009). Our data revealedthat transcript levels of OsNCED2, encoding one of therate-limiting enzymes in ABA biosynthesis, decreased,and OsABA8ox1 and OsABA8ox2 encoding ABA inac-tivation enzymes were up-regulated in both genotypesduring submergence (Fig. 3C), indicating that the de-crease in ABA content may be explained by changes inthe expression levels of genes related to ABA metabo-lism in both genotypes.

JA Is a Novel Regulatory Factor Associated with StemElongation in Deepwater Rice

Kende et al. (1998) reported that ethylene, ABA, andGA are key regulators of internode elongation indeepwater rice. In this study, we demonstrated thatsubmergence regulated the endogenous JA content innode samples of rice and that JA inhibited thesubmergence-induced internode elongation in cvC9285 plants (Fig. 4). CYP94C2b encodes a cytochromeP450 enzyme and was involved in the inactivation ofJA-Ile (Kurotani et al., 2015). Our data showed thatCYP94C4, which is a major expressed CYP94C gene,showed increased expression levels during submer-gence in both genotypes. Thus, the increase in CYP94C4might regulate the decrease in endogenous JA content.The decrease in JA is a common response to submer-gence in both genotypes, like the changes in ethyleneand ABA contents during submergence. The non-deepwater rice cv T65 at 6LS has no ability to elongateits internodes even in the case of GA treatment (Nagaiet al., 2014), suggesting that the changes in JA contentsin cv T65 plants do not affect stem elongation.

Studies of cross talk between JA and GA signalingpathways have shown that GA antagonistically func-tions in JA signaling pathways involved in plantgrowth and development processes in Arabidopsis(Hou et al., 2013; Wasternack and Hause, 2013). In ad-dition, Yang et al. (2012) showed that JA-mediatedgrowth inhibition in rice was caused by changes in thelevels of DELLA repressors and interference with GAsignaling. Our data suggest that GA-mediated inter-node elongation in deepwater rice requires the sup-pression of JA function through the reduction of JAcontent in submergence.

The cv C9285 and T65 Plants Differentially ExpressTrehalose and Fermentation Metabolism-Related Genes

Trehalose, which accumulates in response to vari-ous stresses, acts as a carbon source and an osmopro-tectant by stabilizing proteins and membranesagainst the stresses (Krasensky and Jonak, 2012). On theother hand, trehalose-6-phosphate (Tre6P), a precur-sor of trehalose, functions as a signaling metabolitethat coordinates carbon assimilation, starch synthe-sis, nitrogen metabolism, growth, and development(Schluepmann et al., 2003; Martins et al., 2013; Lunn

et al., 2014; Yadav et al., 2014; Figueroa et al., 2016;Figueroa and Lunn, 2016). The change in the Tre6P-Sucratio is an important homeostatic mechanism of plantsunder stress conditions; thus, Tre6P also acts as a signalfor Suc status through negative feedback regulation ofSuc levels (Yadav et al., 2014; Figueroa and Lunn, 2016).In rice, overexpression of OsTPS1 or OsTPP1 enhancedthe tolerance to abiotic stresses, leading to the expres-sion of stress-related genes (Ge et al., 2008; Li et al.,2011a). OsTPP7 (OsTPP11 in this article) also enhancesthe anaerobic germination tolerance in young rice byregulating the trehalose content and Tre6P/Suc ho-meostasis in sugar signaling, but not the Tre6P content(Kretzschmar et al., 2015). In our data, although sev-eral OsTPS genes, including OsTPS1, OsTPP1, andOsTPP11, were expressed by submergence in bothgenotypes, the expression of OsTPS12, OsTPS13, andOsTPS14was induced dramatically by submergence incv C9285 but maintained at a lower level in cv T65 (Fig.5B). This result implies that trehalose metabolism dur-ing submergence is differentially regulated between cvC9285 and T65 and that trehalose and Tre6P may ac-cumulate more in submerged cv C9285 plants, con-tributing to the ability of deepwater rice to withstandand adapt to submergence stress. In submergedSUB1A-1-containing M202(SUB1) rice seedlings andanoxic coleoptiles, the trehalose biosynthesis pathwayis activated (Jung et al., 2010), suggesting that such apathway also is utilized by the deepwater rice as a partof metabolic adaptation to submergence.

Under low-oxygen conditions, glycolysis is chan-neled predominantly to fermentation pathways (in-stead of aerobic respiration), which is necessary for cellsurvival to produce energy and recycle carbon for otherpathways (Gibbs and Greenway, 2003; Voesenek et al.,2006). Our data exhibited the increased expression offermentation-related genes, PDCs, ADH1 and ADH2,and ALDH2a, during submergence in both genotypes(Fig. 6B). A functional SUB1 locus regulates the geneexpression and enzyme activities of PDC and ADHduring submergence (Fukao et al., 2006). Thus, aSUB1A-1-containing rice, cv Flood Resistant 13A, ac-cumulates less aldehydes because high expression ofADH enhances the detoxification of acetaldehyde intothe neutral and diffusible ethanol (Singh et al., 2001; Xuet al., 2006). Submergence also induces the expressionof ALDH2a, but not ALDH1, in coleoptiles of rice(Nakazono et al., 2000). Our results imply that the fer-mentation pathway is active in both genotypes and thatthe enzymes ADH and ALDH2a are likely involved inremoving toxic alcohol and acetaldehyde in response tolow oxygen during submergence (Fig. 6A).

The increased expression of OsAlaAT1 (Fig. 6B) fur-ther supports that the active fermentation pathway incv C9285 and T65 also generates Ala. These results areconsistent with the increased activities of AlaAT, PDC,and ADH in the flooded coleoptile of rice cv Nippon-bare (Kato-Noguchi, 2006); however, in their study, theactivity of LDH and lactate content were similar both inthe presence and absence of oxygen. In our data set, the


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transcripts of two LDH genes in cv C9285 plants werestrongly up-regulated after submergence comparedwith T65 plants (Fig. 6B). The formation of lactate leadsto a decrease in cytosolic pH, and the initial acidifica-tion of the cytoplasm helps to achieve optimum activityof PDC, which, in turn, promotes the switch from lac-tate to ethanol fermentation (Roberts et al., 1984;Kennedy et al., 1992; Magneschi and Perata, 2009).Hence, it is possible that the abundance of LDH tran-scripts results in higher accumulation of lactate in thecytoplasm and a stronger lactate-dependent reductionof cytosolic pH favoring ethanol fermentation more incv C9285 than in cv T65 plants. Recently, Lee et al.(2015) reported that lactate binds to NDRG3, anoxygen-regulated protein, and activates Raf-ERK sig-naling to promote cell growth and angiogenesis duringhypoxia in human cells, suggesting that lactate in plantcells might function as a signaling molecule in a similarsignaling pathway in response to hypoxia.

Cell Wall Synthesis and Modification Genes AreDifferentially Expressed in cv C9285 Plants in Responseto Submergence

Genes with a differential response to submergencebetween cv C9285 and T65 plants were enriched in thecell wall-related category (Supplemental Fig. S2C).Additionally, genes encoding cell wall-related proteins,such as EXPs, XTHs, PMEs, and structural cell wallproteins (FLAs and extensins), were categorized assubmergence-induced genes in both genotypes(Supplemental Table S4, A and B) and showed higherexpression in cv C9285 plants under the nonsubmergedcondition (Fig. 7A). Elongating cells require the relax-ation of cell walls, involving cell wall-loosening factorssuch as EXPs. EXP activity increases under acidic con-ditions, which are controlled by plasma membraneH+-ATPase activity via auxin-induced SMALL AUXINUP-RNA proteins (Perrot-Rechenmann, 2010; Spartzet al., 2014). In deepwater rice, a positive correlationwas observed between the expression of EXPs and acid-induced cell wall extensibility, and theymay function ininternode elongation (Cho and Kende, 1997a, 1997b;Lee and Kende, 2001). The EXPs together with XTHscontrol fiber orientation and viscoelastic properties ofthe matrix facilitating cell wall expansions (Cosgrove,2000; Van Sandt et al., 2007). XTH activity possiblyenhances EXP activity during the shade-avoidanceelongation response in Stellaria longipes (Sasidharanet al., 2008). In rice, XTHs could function in the cell wallformation of the vascular bundles in elongating stems(Hara et al., 2014).PMEs catalyze the demethylesterification of pectin, a

GalUA-rich cell wall polymer, resulting in the forma-tion of a gel-like structure of polymers and thereby in-creasing wall porosity and extension of cells (Jolie et al.,2010). Jeong et al. (2015) showed that high PME activitywas detected in germinating shoots that actively un-dergo cell elongation. The cell wall also is regulated by

structural cell wall proteins, such as the arabinoga-lactan proteins, which are involved in elongation andgrowth of Arabidopsis and cucumber (Cucumis sativus;van Hengel and Roberts, 2002; Park et al., 2003). Wefound that that the expression of several genes relatedto cell wall loosening was induced. These genes mightcontribute to cell wall reconstruction and increased cellwall extensibility, leading to further enhancement ofrapid internode elongation of cv C9285 plants duringsubmergence.

The accumulation of lignin is part of the cell wallmodifications that lead to an increase in mechanicalstrength but also inhibit cell elongation. In cv C9285plants, the expression of several CAD genes, encodingkey enzymes that catalyze the final step in monolignolsynthesis, decreased during submergence (Fig. 7B),leading to a reduced lignin content in newly elongatedinternodes during submergence (Fig. 7E).

Peroxidase genes showed differential expressionpatterns between both genotypes under submergence(Supplemental Fig. S2C; Supplemental Results S1)and were classified as submergence-induced genes(Supplemental Table S4, A and B). Interestingly, theexpression levels of the peroxidase genes were higher incv C9285 plants than in cv T65 plants during the earlysubmergence condition (Supplemental Fig. S6C). Per-oxidases are involved in lignification, stress defense,and the regulation of reactive oxygen species produc-tion (Shigeto and Tsutsumi, 2016). In addition, hy-droxyl radicals produced by peroxidases also mayfunction in cell wall loosening and cell elongation, as inmaize (Zea mays) roots (Liszkay et al., 2004). Thus,submergence-responsive peroxidases probably func-tion in cell wall loosening and the lignification ofelongating internodes in cv C9285 plants.

ERF Genes Are Differentially Regulated in Submerged cvC9285 and T65 Plants

ERFs are major downstream components of the eth-ylene signaling pathway. In our analysis, among the115 detected ERF family transcripts, 39 genes in the Ib,IIa, IIIc, VII, VIIIa, IXa, X, and XI subgroups wereup-regulated and 10 genes in the IIIe and Va subgroupswere down-regulated by submergence in cv C9285plants (Supplemental Table S5), indicating that ethyl-ene signaling is an important regulatory pathway forthe response of deepwater rice to submergence.

Interestingly, among all 15 members of the group VIIERF family, 12 genes, including SUB1B (OsERF#063)and SUB1C (OsERF#073), were induced by submer-gence in cv C9285 plants (Supplemental Table S5;Supplemental Fig. S8A). Members of the group VII ERFfamily act as oxygen sensors under hypoxic conditionsby the N-end rule pathway for protein degradation inArabidopsis (Gibbs et al., 2011; Licausi et al., 2011;Gasch et al., 2016) and function in multiple stress tol-erances, such as drought, cold, pathogen attack, salin-ity, osmotic stress, and submergence, in various plants














(Licausi et al., 2010; Mizoi et al., 2013; Gibbs et al., 2015;Papdi et al., 2015). We found that the other three genes(OsERF#70, OsERF#71, and OsERF#72) encoding aCMVII-4 motif, which is predicted to be a phospho-rylation site of mitogen-activated protein kinases(MPKs; Nakano et al., 2006), showed nonresponsiveexpression to submergence in cv C9285 plants(Supplemental Fig. S13). MPKs phosphorylate a largenumber of transcription factors, including ERFs(Popescu et al., 2009), and the MPK-induced phospho-rylations in rice ERFs enhance their transcriptional ac-tivities and environmental stress tolerances (Cheonget al., 2003; Schmidt et al., 2013). Recently, it wasreported that OsMPK3 phosphorylated SUB1A-1 tocontrol shoot elongation in a SUB1A-1-dependentmanner, although the phosphorylation site was notlocated in the CMVII-4 motif (Singh and Sinha, 2016). Incv C9285 plants, all members of the group VII ERFs thatencode proteins without the phosphorylation sites wereinduced by submergence, and the submergence-induced expression levels of some group VII ERFs(OsERF#59 , OsERF#61 , OsERF#63 , OsERF#64 ,OsERF#66, and OsERF#67) were higher during sub-mergence compared with cv T65 plants (SupplementalFig. S13). These results suggest that phosphorylation-independent group VII ERFs may be important for thesubmergence-induced stress response in deepwaterrice, although there is still the possibility that submer-gence induces phosphorylation of the ERFs by MPKs.

Other submergence-induced ERFs in subgroupsIIa and VIIIa encode proteins that contain an ERF-associated amphiphilic repression (EAR) motif(DLNxxP), which acts as a transcriptional repressordomain (Fujimoto et al., 2000; Ohta et al., 2001). AsEAR-containing transcription factors regulate variousstress responses (Kazan, 2006; Dong and Liu, 2010;Licausi et al., 2013), the submergence-induced ERFsmight be involved in negatively regulating the expres-sion of dispensable genes for the submergence re-sponse.

In our data, group IIIc, IXa, and X ERFs alsoshowed up-regulated expression in response to sub-mergence (Supplemental Table S5). In Arabidopsis,the group IIIc, IXa, and X ERFs function in toleranceto environmental stresses such as freezing, salt, anddehydration (e.g. cold-binding factor/dehydration-responsive element binding1 protein; Dubouzetet al., 2003), the ethylene-JA signaling pathway(Lorenzo et al., 2003; Champion et al., 2009), and theresponse to ABA and various stresses (Pandey et al.,2005; Zhu et al., 2010), respectively. In the case of thegroup XI ERFs, this family exists in rice but not inArabidopsis, indicating that these genes have rice-specific functions.

A group IIIe ERF, TINY, functions in the reduction ofcell expansion and differentiation, leading to a dwarfphenotype in Arabidopsis (Wilson et al., 1996). In cvC9285 plants, two group IIIe ERF family genes weredown-regulated by submergence, suggesting that thegroup IIIe ERFs possibly affect cell expansion during

submergence. Arabidopsis ERF11 in group VIIIarepresses ethylene biosynthesis genes but promotes cellelongation by promoting GA accumulation and inhib-iting DELLA function (Li et al., 2011b; Zhou et al., 2016).In rice, OsEATB (OsERF#102) in group Xb and OsERF3(OsERF#075) in group VIIIa negatively regulate inter-node elongation (Qi et al., 2011; Zhang et al., 2013). In cvC9285 plants, OsEATB was not expressed and OsERF3was not differentially expressed during submergence(Supplemental Fig. S11D), suggesting that the twonegative regulators do not participate in GA-mediatedinternode elongation during submergence in cv C9285plants.

SUB1A up-regulates the transcriptional expressionlevel of 12 ERF family genes (Jung et al., 2010). Al-though the SUB1A gene is not present in the cv C9285genome (Hattori et al., 2009), the six SUB1A-dependentexpressed genes (i.e. OsERF#025, OsERF#066,OsERF#067, OsERF#068, OsERF#076, and OsERF#077)also were induced by submergence in cv C9285 plants(Supplemental Fig. S8A). All SUB1A downstream ERFgenes belong to the IIIc, VIIa, and VIIIa subfamilies.This indicates that these genes also are regulated byother factors in cv C9285 plants and may indicate thattheir expression is not associated directly with inter-node elongation in deepwater rice.

The ERF transcription factors bind to the promoterregions of their target genes, such as pathogen-relatedgenes, through the recognition of a GCC-box cis-regulatory sequence (GCCGCC [core motif]; Ohme-Takagi and Shinshi, 1995; Hao et al., 1998; Solanoet al., 1998). Our in silico analysis showed that thetwo GCC-box-like motifs (G/CCGGCGGCGG andCGCCGCCGCC) were enriched in the promoter re-gions of genes with higher expression in cv C9285plants (Supplemental Fig. S9), suggesting that thesegenes with GCC-box-like motifs are regulated prefer-entially by ERFs. Some submergence-induced ERFgenes showed higher expression in cv C9285 than thoseof cv T65 plants before and after submergence(Supplemental Fig. S8A). This result suggests that theERF genes in cv C9285 are associated with the consti-tutively higher expression of their putative down-stream target genes. Recently, Gasch et al. (2016)identified a novel hypoxia-responsive promoter ele-ment that is a binding site of ERF VIIs, RELATED TOAP2 2 (RAP2.2) and RAP2.12, the key regulators of theN-end rule pathway in Arabidopsis. However, we didnot identify the hypoxia-responsive promoter elementmotif in our promoter analysis of cv C9285-enrichedgenes. The oxygen levels in underwater nodes couldbe much higher than expected for two reasons: (1) dif-fusion from the water layer into the node, and (2) un-derwater photosynthesis. Under submergence withlight illumination, the oxygen concentration of deep-water rice initially dropped but recovered within90 min by underwater photosynthesis (Stünzi andKende, 1989). Thus, our data may not be suitable tostudy hypoxia, since we collected all samples underlight conditions.


Minami et al.











A Model for the Transcriptional Response of DeepwaterRice to Submergence

We propose a model for submergence responsesduring the escape strategy in the deepwater rice cvC9285 (Supplemental Fig. S14). Submergence subjectsplant cells to multiple stresses, such as limitation of gasdiffusion, lower light intensity, high risk of pathogeninfection, and decreased oxygen uptake. To avoid crit-ical damage from submergence, cv C9285 plants haveevolved to rapidly elongate their internodes and leaves.We revealed that changes in the contents of the planthormones ethylene, ABA, GA, and JA during submer-gence were transcriptionally regulated. Ethylene sig-naling leads to biotic and abiotic stress tolerances bychanging the expression of many genes and plant hor-mone levels (Müller and Munné-Bosch, 2015), and ourdata largely exhibited changes in the putative down-stream components of ethylene signaling during sub-mergence.We also proposed that JA is a novel regulatorfor submergence-induced internode elongation in cvC9285 plants. In deepwater rice, GA has an importantrole in the elongation response, because GA treatmentinduces the expression of cell wall-related genes such asEXPs and changes the activity of CAD (Sauter andKende, 1992; Cho and Kende 1997c). We found that cvC9285-specific accumulation of active GAs was proba-bly controlled by the expression of GA20ox2 duringsubmergence (Fig. 3B), and the cell wall-related genesshowed higher expression in cv C9285 plants (Fig. 7A).Furthermore, the reduced lignin content in cv C9285plants also may facilitate the elongation of internodesunder the submerged condition. Overall modificationsin cell wall metabolism also may contribute to the rapidinternode elongation of deepwater rice. Among thetranscription factor genes, the ERF family genes wereexpressed especially at high levels during submergencein cv C9285 plants. Interestingly, genes expressed spe-cifically in cv C9285 plants contain the GCC-box-likemotifs that are recognized by ERFs. This indicatesthat ethylene signaling and the transcriptional responsepathway via ERFs could be key factors for the sub-mergence response in cv C9285 plants. In fact, riceETHYLENE INSENSITIVE3-LIKE1, which is the mas-ter regulator of ethylene signaling, binds to the pro-moter region of the ethylene/submergence-inducedERF transcription factors SK1/2 that positively, but notexclusively, regulate internode elongation in cv C9285plants (Hattori et al., 2009). Since we could not find anycv C9285-specific novel submergence-induced tran-scription factors in the cv C9285 genome by our de novoassembly analysis and the presence of SK1/2 alonedoes not lead to a full stem elongation response, addi-tional transcription factors in coordination with SK1/2are most likely involved.In R. palustris, genes associated with photomorpho-

genesis and shade avoidance seem to regulate the un-derwater elongation response (van Veen et al., 2013). Inour data, the expression pattern of light signaling-regulated genes in cv C9285 plants did not show any

obvious genotype or submergence-specific pattern(Supplemental Fig. S15). The genes involved in fer-mentation and trehalose metabolic pathways respon-ded to submergence in both cv C9285 and T65 plants,and we found that TPSs and LDHs were expressedpreferentially during submergence in cv C9285 plants(Figs. 5 and 6), suggesting that thesemetabolic activitiesincrease more strongly in cv C9285 plants during sub-mergence compared with normal rice such as cv T65plants, resulting in adaptation to long-term submer-gence.

Our experiments showed that many pathogenesisrelated proteins were expressed preferentially in eachrice variety (Supplemental Table S3), and several genesencoding disease resistance proteins were identified ascv C9285-unique transcripts (Fig. 8). Waterloggedconditions increase the risk of pathogen infection inplants (Tamang and Fukao, 2015). Accordingly, rice inpaddy fields have protection mechanisms that preventdamage rendering them vulnerable to pathogens (Hsuet al., 2013). Therefore, these cv C9285-specific genesmight function as a pathogen defense pathway duringpartial long-term submergence in cv C9285 plants.

Our studies identified genotype-specific responsivegenes to submergence in cv C9285 plants and assumedthat the putative key genes would be associated withsubmergence-induced physiological responses in cvC9285 plants. Furthermore, we showed that ethylene isone of the main drivers of submergence responses andthat JA is a new negative regulator of submergence-induced internode elongation in cv C9285 plants. Thenetwork involving submergence-induced changes andgenetic factors complexly regulates underwater elon-gation and adaptation in deepwater rice.

MATERIALS AND METHODS

Plant Material and Cultivation

A deepwater rice (Oryza sativa) cultivar (cv C9285) and a nondeepwater ricecultivar (cv T65; japonica) were used. Rice seeds were incubated at 60°C for10 min followed by pregermination at 29°C in water for 3 to 4 d. Afterward,germinated seeds were transferred to plastic pots containing soil mixture(Mikawa Baido; AICHI Mederu) and grown in a greenhouse in Nagoya, Japan,in Junewith a natural light cycle of approximately 14 h of light/10 h of dark. Fordeepwater treatment, rice seedlings that reached the indicated leaf stages (4LSand 6LS) were completely submerged for 1, 3, 6, 12, and 24 h (Supplemental Fig.S1A). To avoid differences in gene expression between samples due to circadianrhythms, submergence treatments were initiated at different times of the dayand all samples were collected in the afternoon, although varying durations ofunderwater and abovewater photosynthesis cause changes in the availability ofphotosynthates. After submergence treatment, 5 mm of the shoot base regioncontaining internodes, nodes, the shoot apex, and basal regions of leaves wassampled, rapidly frozen in liquid nitrogen, and stored at 280°C until RNAextraction (Supplemental Fig. S1B).

RNA Extraction, Sequencing, and Read Mapping

For each RNA extraction, frozen tissues from one individual plant werehomogenizedandupto100mgwasused.TotalRNAofeachsamplewas isolatedusing the RNeasy Plant Mini Kit (Qiagen) with the RNase-Free DNase Set(Qiagen). RNA purity was checked using a NanoDrop spectrophotometer(Thermo-Fisher), and RNA was quantified using the QuantiFluor RNA system











(Promega) and the EnSpire Multimode Plate Reader (PerkinElmer). For HiSeqlibrary construction, 2 mg of total RNA was used with the TruSeq RNA SamplePreparation Kit version 2 (Illumina) according to the manufacturer’s instruc-tions. Agencourt AMPure XP beads (Beckman Coulter) were used to removesmall DNA fragments. The clustering of index-coded samples was performedon the cBot Cluster Generation System using the TruSeq SR Cluster Kit v2-cBot-GA (Illumia) and the TruSeq SBS Kit v5-GA (Illumia). After cluster gen-eration, the library preparations were sequenced on the Illumina GenomeAnalyzer IIx, and 36-bp single-ended reads were generated. Reads weremapped to the IRGSP-1.0 reference transcripts of japonica cv Nipponbare (Goffet al., 2002) using bowtie (version 0.12.7; Langmead et al., 2009) with the -all-best -strata options (Supplemental Table S1), and read counts were quantifiedusing PostgreSQL and a custom PHP script.

Data Analysis

Statistical data analysis was performed using R. The edgeR package was used tonormalize rawcount data and generate cpmvalues (Robinson et al., 2010). Transcriptswith less than 10 averaged counts in at least one condition were removed from thedata set, and only the strongest expressed isoformper transcriptwasused for analysis.To determine differentially expressed genes, a negative binominal generalized log-linear model was used (function glmFit) with each genotype and time point aftersubmergence, defined as one group. Then, likelihood ratio tests (function glmLRT)were performed to compare between groups or to test for interactions between gen-otype and submergence treatment. The Bonferroni-Holmmethodwas used to correctfor multiple testing (function topTags). PCA was performed using the prcomp func-tion with scaled and centered cpm data as the input. The k-means clustering wasperformed using theMBClusterSeq package with a negative binomial model and theEM algorithm (Si et al., 2014). The number of clusters was varied between 10 and100 in intervals of 10, and empirically k = 40 (for all samples) or k = 20 (for samplesfrom one genotype only) was chosen as the most appropriate number of clusters.Hierarchical clustering of transcription factor genes was performed using the dist andhclust functions. The distance matrix was calculated using Euclidian distance, andclustering was performed using the average algorithm.

Gene set enrichment analysis was based on the MAPMAN ontology, andmappings (Rice Annotation Project Database locus identifier to MAPMAN bin)were obtained from http://www.mapman.gabipd.org (Ramsak et al., 2014).Significant enrichment of bins was determined using Fisher’s exact test, andcorrections for multiple testing were done by the Bonferroni-Holm methodusing the R functions fisher.test and p.adjust, respectively. Separate analyseswere performed for the first three hierarchy levels of the MAPMAN ontology.

Metabolic maps were based on a combination of Rice Annotation ProjectDatabase Kyoto Encyclopedia of Genes and Genomes (Kanehisa et al., 2016),MAPMAN, and in-house annotations. Data visualization was performed usingggplot2 (Wickham, 2009).

ABA and GA3 Treatments of Whole Plants

Germinated seeds of cvC9285plantswere sown inplastic potsfilledwith soiland then grown in a phytotron at 25°Cwith a 14-hphotoperiod.When the plantswere grown at the 4LS, they were transferred to new plastic containers forhormone treatment. For hormone treatment, plants were watered with 10 mM

ABA (Sigma-Aldrich), 10 mM GA3 (Wako), or a combination of both into thecontainers. The water level in each container was controlled to keep the hor-mone concentration constant during the treatment. Plant height and internodelength were measured after 18 d of treatment. Internode length was calculatedusing the total length of all internodes.

JA Treatment of Whole Plants during Submergence

Germinated seeds of cvC9285plantswere sown inplastic potsfilledwith soiland then grown in a controlled-environment chamber at 25°C with a 14-hphotoperiod. When the plants reached the 6LS, they were submerged in waterwith or without 50 mM methyl jasmonate (Wako) for 3 d. Total internode lengthwas calculated as the sum of the length of all internodes.

Measurement of Plant Hormone Contents

The concentrations of endogenous hormones were measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-XevoTQ-S; Waters) as described by Kojima et al. (2009).

Measurement of Lignin Content

Nodes and internodeswere sampled from cvC9285 andT65 plants at the 6LSor 7LS before and after submergence treatment for 2 d. Lignin content wasdetermined by the thioglycolic acid method according to Suzuki et al. (2009).

Promoter Motif Enrichment Analysis

Transcripts were clustered into six groups based on the expression in cvC9285 and T65 plants (6LS) at 0, 1, 3, 6, 12, and 24 h after submergence usingk-means clustering as described above. For tentative construction of a cv C9285genome, all reads obtained from whole-genome sequencing of cv C9285 weremapped against the IRGSP-1.0 pseudomolecules using bwa-mem with the -Moption (Li and Durbin, 2009). Mapped reads were realigned using Realign-erTargetCreator and indelRealigner from the GATK software suite (DePristoet al., 2011). To identify single-nucleotide polymorphisms and insertions/deletions, UnifiedGenotyper of GATK was used with the -glm BOTH option.The cv C9285 genome sequence was tentatively constructed by modifying theIRGSP-1.0 genome with the identified variants of cv C9285 by custom Perlscripts. DNA sequences 1 kb upstream of the translational start sites from the cvC9285 genome were regarded as promoter regions and used for motif enrich-ment analysis. CENSOR was used to mask low-complexity regions in thepromoter sequences (Kohany et al., 2006). The MEME suite was used to findenriched DNA sequence motifs using the following parameters: -dna -nmotifs10 -maxw 10 (Bailey and Elkan, 1994).

Accession Numbers

All readsused in this study canbe found in theDNADatabase of Japanunderbioproject PRJDB5294 (cv C9285 RNA-Seq reads) and bioproject PRJDB5300(cv C9285 genomic reads).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic overview of the experimental setupand transcriptome sequencing.

Supplemental Figure S2. Significance analysis using a generalized log-linear model.

Supplemental Figure S3. Expression of genes related to ethylene, GA, andABA signaling during submergence.

Supplemental Figure S4. Overview of MAPMAN visualization of differ-entially expressed genes in cv C9285 plants after 1 h of submergence.

Supplemental Figure S5. Overview of MAPMAN visualization of differ-entially expressed genes in cv C9285 and T65 plants under the nonsub-merged condition.

Supplemental Figure S6. Expression of lignin biosynthesis genes duringsubmergence.

Supplemental Figure S7. Clustering of transcription factor genes accord-ing to expression in cv C9285 relative to cv T65 plants.

Supplemental Figure S8. Submergence-induced AP2/EREBP family genesin cv C9285 plants.

Supplemental Figure S9. Enriched sequence motifs in the promoters ofgenes that are differentially expressed in cv C9285 plants.

Supplemental Figure S10. Analysis pipeline to detect novel cv C9285 tran-scripts.

Supplemental Figure S11. Submergence responses related to plant hor-mones.

Supplemental Figure S12. Isolation of genes specifically induced by sub-mergence in cv C9285 4LS plants.

Supplemental Figure S13. Expression of group VII ERF genes during sub-mergence.

Supplemental Figure S14. Molecular model for the submergence responsein the deepwater rice cv C9285.


Minami et al.



http://www.mapman.gabipd.org
















Supplemental Figure S15. Expression of light signaling genes during sub-mergence.

Supplemental Table S1. Summary of read mapping statistics.

Supplemental Table S2. Number of genes with significantly different ex-pression in pairwise comparisons.

Supplemental Table S3. Enriched MAPMAN bins among genes preferen-tially expressed in cv C9285 or T65 plants.

Supplemental Table S4. Enriched MAPMAN bins among genes inducedor repressed by submergence in either genotype.

Supplemental Table S5. Response to submergence of rice ERF subfamilygenes in cv C9285 plants.

Supplemental Methods S1.

Supplemental Results S1.

Supplemental Data Set S1.














ACKNOWLEDGMENTS

We thank Dr. Ko Hirano for assisting with lignin quantification andDr. M. Fujita for providing cv C9285 genomic sequencing data. Seeds of cvC9285 used in this study were distributed from the National Institute ofGenetics supported by the National Bioresource Project, AMED, Japan.

Received July 7, 2017; accepted February 15, 2018; published February 23, 2018.

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