An NADPH Oxidase RBOH Functions in RiceRoots during Lysigenous Aerenchyma Formation underOxygen-Deficient Conditions

Takaki Yamauchi,a Miki Yoshioka,a Aya Fukazawa,a Hitoshi Mori,a Naoko K. Nishizawa,b,c Nobuhiro Tsutsumi,b

Hirofumi Yoshioka,a and Mikio Nakazonoa,d,1

aGraduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, JapanbGraduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, JapancResearch Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa 921-8836, JapandUWA School of Agriculture and Environment, Faculty of Science, The University of Western Australia, Crawley WA 6009, Australia

ORCID IDs: 0000-0002-6772-6506 (T.Y.); 0000-0001-7331-0884 (H.M.); 0000-0002-2610-6837 (N.T.); 0000-0003-3956-7250 (H.Y.);0000-0001-7119-2052 (M.N.)

Reactive oxygen species (ROS) produced by the NADPH oxidase, respiratory burst oxidase homolog (RBOH), trigger signaltransduction in diverse biological processes in plants. However, the functions of RBOH homologs in rice (Oryza sativa) andother gramineous plants are poorly understood. Ethylene induces the formation of lysigenous aerenchyma, which consistsof internal gas spaces created by programmed cell death of cortical cells, in roots of gramineous plants under oxygen-deficient conditions. Here, we report that, in rice, one RBOH isoform (RBOHH) has a role in ethylene-induced aerenchymaformation in roots. Induction of RBOHH expression under oxygen-deficient conditions was greater in cortical cells than incells of other root tissues. In addition, genes encoding group I calcium-dependent protein kinases (CDPK5 and CDPK13)were strongly expressed in root cortical cells. Coexpression of RBOHH with CDPK5 or CDPK13 induced ROS production inNicotiana benthamiana leaves. Inhibitors of RBOH activity or cytosolic calcium influx suppressed ethylene-inducedaerenchyma formation. Moreover, knockout of RBOHH by CRISPR/Cas9 reduced ROS accumulation and inducibleaerenchyma formation in rice roots. These results suggest that RBOHH-mediated ROS production, which is stimulatedby CDPK5 and/or CDPK13, is essential for ethylene-induced aerenchyma formation in rice roots under oxygen-deficientconditions.

INTRODUCTION

Plants have no active dispersal mechanisms for transportingoxygen; thus, internal transport of oxygen is dominated by dif-fusion (Armstrong and Armstrong, 2014). To adapt to water-logging in soil, some gramineous plants form lysigenousaerenchyma (internal gas spaces) in roots as a result of cell deathand lysis of cortical cells (Jackson and Armstrong, 1999; Evans,2003). Internal oxygen transport from shoots to roots throughlysigenousaerenchyma isessential for survival inwaterloggedsoil(Armstrong, 1979; Colmer, 2003). In rice (Oryza sativa), lysigenousaerenchyma is constitutively formed under aerobic (well-drained)conditions (Jackson et al., 1985; Shiono et al., 2011), and itsformation is further inducedunder oxygen-deficient (waterlogged)conditions (Justin and Armstrong, 1991; Shiono et al., 2011;Colmer and Voesenek, 2009). These processes are called con-stitutive and inducible aerenchyma formation, respectively.

Programmed cell death (PCD) is involved in diverse de-velopmental processes and biotic/abiotic stress responses inplants (Van Hautegem et al., 2015). Several lines of evidence

(reviewed in Evans, 2003) show that cell collapse during lysige-nous aerenchyma formation resembles the canonical apoptoticpathway in animal cells. Under waterlogged conditions, thegaseous phytohormone ethylene accumulates in roots due toethylene biosynthesis and its low diffusion rate to the rhizosphere(Sasidharan and Voesenek, 2015). Upon accumulation, ethylenestimulates the PCD that occurs during lysigenous aeren-chyma formation (Justin and Armstrong, 1991; Colmer et al.,2006). Ethylene is biosynthesized by two main successiveenzymatic reactions: conversion of S-adenosylmethionine to1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase(ACS), followedby conversion of ACC to ethylenebyACCoxidase(ACO; Yang and Hoffman, 1984). The rice genome has six genesencoding ACSs and seven genes encoding ACOs (Iwai et al.,2006; Yamauchi et al., 2016). During ethylene-dependent in-ducible aerenchyma formation in rice roots under oxygen-deficient conditions, the expression of ACS1 and ACO5 is greatlyincreased (Yamauchi et al., 2015). The reduced culm number1(rcn1) mutant has amutation in the gene encoding the ATP bindingcassette (ABC) transporter RCN1/OsABCG5 (Yasuno et al., 2009).The expression of ACS1 is reduced in rcn1 roots, which reducesthe amounts of ethylene and aerenchyma that are formed underoxygen-deficient conditions (Yamauchi et al., 2015).Reactive oxygen species (ROS), such as superoxide anion

radical (O2$2) and hydrogen peroxide (H2O2), are essential in

several types of signal transduction in plants (Mittler et al., 2011).

1 Address correspondence to nakazono@agr.nagoya-u.ac.jp.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theinstructions for Authors (www.plantcell.org) is: Mikio Nakazono(nakazono@agr.nagoya-u.ac.jp).www.plantcell.org/cgi/doi/10.1105/tpc.16.00976

The Plant Cell, Vol. 29: 775–790, April 2017, www.plantcell.org ã 2017 ASPB.

ROS signaling has a key role in the acclimation of plants to low-oxygen conditions (Bailey-Serres and Voesenek, 2008; Sauter,2013; Steffens et al., 2013; Pucciariello and Perata, 2017).Lysigenousaerenchyma formation in rootsofmaize (Zeamayssspmays; Rajhi et al., 2011) and wheat (Triticum aestivum; Yamauchiet al., 2014) is regulated through ROS signaling. Rice formslysigenous aerenchyma in shoots (Colmer and Pedersen, 2008),where ROS regulate its formation (Parlanti et al., 2011; Steffenset al., 2011).

Respiratory burst oxidase homolog (RBOH), a plant homolog ofmammalian NADPH oxidase, converts O2 to O2

$2 (Torres andDangl, 2005). Plant RBOHs constitute a multigene family (Suzukiet al., 2011). The Arabidopsis and rice genomes have 10 and9 RBOH genes, respectively (Torres and Dangl, 2005; Wonget al., 2007), andeach homologhas a specific role in abroad rangeofbiologicalprocesses (Marinoetal.,2012).For instance,At-RBOHBregulates seed ripening (Müller et al., 2009), At-RBOHC reg-ulates root hair formation (Foreman et al., 2003), At-RBOHEregulatesPCD in the tapetum (Xieet al., 2014), andAt-RBOHDandAt-RBOHF regulate the immune response (Torres et al., 2002,2005). By contrast, some RBOHs mediate multiple functions(Suzuki et al., 2011). For example, At-RBOHD mediates ROSproduction in the immune response and stomatal closure (Zhanget al., 2009), as well as in systemic signal transduction duringresponses to external stimuli (Miller et al., 2009) and in ligninproduction (Denness et al., 2011). Moreover, the survival rate ofseedlings under anoxic (anaerobic) conditions is reduced in therbohD mutant in Arabidopsis thaliana (Pucciariello et al., 2012),suggesting that At-RBOHD also mediates signal transductionunder anoxic conditions. In rice, Os-RBOHA and Os-RBOHE(Yoshie et al., 2005) aswell asOs-RBOHB (Nagano et al. 2016) areinvolved in immune responses. In maize, ROOTHAIRLESS5,which is a monocot-specific NADPH oxidase, regulates roothair formation (Nestler et al., 2014). However, the functions ofother RBOHs in rice and in other gramineous plants are poorlyunderstood.

In maize roots under hypoxic (oxygen-deficient) conditions,expression of genes encoding the A, B, C, and D isoforms ofZm-RBOH is stimulated during the cell death of the apical meri-stem (Mira et al., 2016). Previously, we found that waterlogginginduces Zm-RBOHH expression in the cortical cells of primaryroots at the same time it induces aerenchyma formation (Rajhiet al., 2011). Moreover, aerenchyma formation is blocked by DPI(diphenyleneiodonium), an NADPH oxidase inhibitor (Yamauchiet al., 2011). These findings suggest that RBOH-mediated ROSproduction is essential for inducible aerenchyma formation inroots of maize and other gramineous plants.

Some RBOH proteins are activated via direct phosphorylationby calcium (Ca2+)-dependent protein kinases (CDPKs/CPKs),which are Ser/Thr protein kinases activated by Ca2+ (BoudsocqandSheen, 2013;Adachi andYoshioka, 2015;Kurusuetal., 2015).CDPK consists of an N-terminal variable (V), protein kinase (K),junction, and calmodulin-like domains (Harper et al., 2004). PlantCDPKs can be classified into four groups (I, II, III, and IV) based ontheir amino acid sequences (Cheng et al., 2002). The Arabidopsisand rice genomes have 10 and 11 group I CDPK genes, re-spectively (Cheng et al., 2002; Asano et al., 2005). Some group ICDPKs directly phosphorylate RBOHs (Kobayashi et al., 2007;

Asai et al., 2013; Dubiella et al., 2013; Kadota et al., 2014). Potato(Solanum tuberosum) St-CDPK4 and St-CDPK5 phosphorylatetheN terminus of St-RBOHB in a calcium-dependentmanner, andthese modifications are essential for St-RBOHB-mediated ROSproduction and the immune response (Kobayashi et al., 2007,2012). Inmaize roots,Ca2+-dependent signaling is proposed tobeinvolved in aerenchyma formation (Drew et al., 2000). Indeed,aerenchyma formation in maize roots is blocked by EGTA or ru-thenium red, both ofwhich reduce the cytosolic freeCa2+ level (Heet al., 1996). Ca2+-dependent activation of RBOHs is hypothe-sized to regulate aerenchyma formation (Voesenek and Bailey-Serres, 2015), although it is unclear which RBOH isoforms areinvolved.The objectives of this study were to identify RBOH genes that

are involved in inducible aerenchyma formation in rice roots and tounderstand the molecular mechanisms underlying the Ca2+ andROS signaling events that coordinately regulate it. To this end, weanalyzed the expression patterns of RBOH and group I CDPKgenes in rice roots under aerated conditions and under stagnantconditions (which mimic the changes in gas composition in wa-terlogged soil). Three genes (RBOHH,CDPK5, andCDPK13) werefound tobemost strongly expressed in the root cortical cells understagnant conditions and thus were further examined for roles inaerenchyma formation in rice roots. Activation of RBOHH throughgroup ICDPKs (e.g.,CDPK5andCDPK13)was testedby transientcoexpression, followed by ROS detection in Nicotiana ben-thamiana leaves. Finally, ROS production and aerenchyma for-mation in roots were evaluated in RBOHH knockout lines. Fromthe resultsof thesestudies,weproposeamodel inwhich inducibleaerenchyma formation in rice roots is stimulated by RBOHH-mediated ROS production.

RESULTS

Ethylene-Dependent Aerenchyma Formation in Rice Roots

To investigate the time course of aerenchyma formation in riceadventitious roots, 10-d-old aerobically grown rice (cv Shiokari)seedlingswere transferred to aerated or stagnant (deoxygenated)conditions. After the treatments, transverse sections at 10 mmfrom the tips of adventitious roots were prepared (Figure 1A).Under stagnant conditions, aerenchyma formation startedat 24 to36 h, and peaked at 48 h,whereas under aerated conditions it washardly detected (Figure 1B). At 20 mm from the root tips, someaerenchyma was detected even under aerated conditions, whileunder stagnant conditions induction of aerenchyma formationstarted at 24 to 36 h (Supplemental Figure 1).The expression of the ethylene biosynthetic gene ACS1 at

10 mm (62 mm) from the tips of adventitious roots started toincrease within 12 h and peaked at 36 h (Figure 1C) and that ofACO5 peaked at 12 h under stagnant conditions (Figure 1D).The patterns of aerenchyma formation in adventitious roots of

20-d-old seedlings were very similar to those of 10-d-old seed-lings (Supplemental Figures 2A and 2B). At 24 h, the content ofethylene in the roots was significantly higher under stagnantconditions than that under aerated conditions (SupplementalFigure 2C).

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Expression of RBOH Genes during Aerenchyma Formation

To identify RBOH genes that are highly expressed during inducibleaerenchyma formation, we measured the absolute transcript levelsofRBOHgenesat 10mm(62mm) from the tipsof adventitious rootsof 10-d-old rice seedlings. Among the nine rice RBOH genes(Supplemental Figure 3 and Supplemental File 1), RBOHA andRBOHH had the highest expression levels at 36 h under stagnantconditions (Figure 2A). In a time-course analysis, the expression ofRBOHA started to increase and peaked at 24 h under stagnantconditions (Figure2B).RBOHHexpressionstarted to increaseat24handgreatly increased (;10-fold)at36h (Figure2C).To investigate thetissue specificities of RBOHA and RBOHH expression, the centralcylinder, cortex, and outer part of the roots were collected fromsectionsat10mm(62mm)fromthetipsofadventitious rootsby lasermicrodissection (Figure 2D). At 36 h under stagnant conditions, theexpressionofRBOHAwashighest at theouterpart of the root (Figure2E), whereas that of RBOHH was highest at the cortex (Figure 2F).

Accumulation of ROS during Aerenchyma Formation

To examine whether RBOH-mediated ROS production is asso-ciatedwith aerenchyma formation,H2O2 contentwasmeasured in

the apical regions (i.e., in segments at 5 to 25mm from the tips) ofadventitious roots of 10-d-old rice seedlings grown under aeratedor stagnant conditions. The content of H2O2 first increased at 24 hand peaked at 36 h under stagnant conditions (Figure 3A).To measure O2

$2 accumulation in roots, adventitious roots at10mmwere stainedwith nitro blue tetrazolium (NBT) at 36 h underaerated or stagnant conditions. The cortex consists of seven(or eight) cell layers (Figure 3B). Under stagnant conditions, cellcollapse and NBT staining were frequently detected at layer 5 or6and thenexpanded radially toadjacentcells (Figure3B). It shouldbe noted that NBT staining was also detected in the outer part ofthe roots under both aerated and stagnant conditions (Figure 3B).The numbers of collapsed and NBT-stained cells peaked at layer5 (Figures 3C and 3D), further supporting the correlation of cellcollapse with O2

$2 accumulation.

Involvements of ROS and Ca2+ Signaling inAerenchyma Formation

AnNADPHoxidase (i.e., RBOH) inhibitor (DPI; 0, 0.05, and0.25mM)dose-dependently inhibited aerenchyma formation at 10 mmfrom the tips of adventitious roots of 10-d-old rice seedlingsgrown under stagnant conditions for 48 h (Figure 4A). DPI alsosuppressed the increased H2O2 content in the apical regions (at5–25mm) of adventitious roots at 36 h under stagnant conditions(Figure 4B).To examine whether Ca2+ signaling is also involved in aeren-

chyma formation in rice roots, 10-d-old aerobically grown riceseedlings were treated with EGTA (0, 50, and 100 mM), which is aninhibitor of cytosolicCa2+ influx from the apoplast, under stagnantconditions for 48 h. EGTA dose-dependently inhibited aeren-chyma formation at 10mm (Figure 4C). EGTAalso suppressed theincreased H2O2 content in the apical regions (at 5–25 mm) ofadventitious roots at 36 h under stagnant conditions (Figure 4D).To further evaluate the source of cytosolic Ca2+ influx, 10-d-old

aerobically grown rice seedlings were treated with ruthenium red(0, 10, and 25 mM), which is an inhibitor of cytosolic Ca2+ releasefrom organelles, under stagnant conditions for 48 h. Rutheniumred dose-dependently inhibited aerenchyma formation at 10 mm(Supplemental Figure 4). Higher concentrations of ruthenium red(10–25 mM) were shown to inhibit cytosolic Ca2+ influx from theapoplast (Subbaiahet al., 1994); thus, suppressionof aerenchymaformation in rice rootsby10or25mMruthenium red treatmentmaybe due to the inhibition of Ca2+ influx from the apoplast.

Expression of CDPK Genes during Aerenchyma Formation

Among the 11 rice group I CDPK genes (Supplemental Figure 5andSupplemental File 2),CDPK5,CDPK7,CDPK10, andCDPK13had the highest expression levels in adventitious roots at 36 hunder stagnant conditions (Figure 5A). In a time-course analysis,the expression ofCDPK5 andCDPK7was increased and peakedat 24 h under stagnant conditions (Figures 5B and 5C), whereasthe expression ofCDPK10was not significantly different betweenaerated and stagnant conditions (Figure 5D).CDPK13 expressionstarted to increase at 24 h and peaked at 36 h (Figure 5E). Theinductions of CDPK5 and CDPK7 expression were earlier thanthe induction of CDPK13 expression under stagnant conditions

Figure 1. Ethylene-Dependent Aerenchyma Formation in Rice Roots.

(A) Cross sections at 10 mm from the tips of adventitious roots of riceseedlings grown under aerated or stagnant conditions. Lysigenous aer-enchyma is indicated by arrowheads. Bars = 100 mm.(B) The percentage of aerenchyma in root-cross-sectional area at 10 mmfrom the root tips under aerated or stagnant conditions.(C)and (D)Time-course relative transcript levelsofACS1 (C)andACO5 (D)at 10mm (62mm) from the root tips under aerated or stagnant conditions.The gene encoding transcription initiation factor IIE, TFIIE, was used asa control.The 20- to 40-mm roots of the 10-d-old aerobically grown rice seedlingswere subjected to the treatments. Values are means 6 SD (n = 9 [B], andn = 3 [C] and [D]). Significant differences between aerated and stagnantconditions at **P < 0.01 and *P < 0.05 (two-sample t test).

RBOHH Regulates Root Aerenchyma Formation 777

(Figures 5B, 5C, and 5E). At 36 h under stagnant conditions, theexpression levels of CDPK5 and CDPK13 were significantly in-creased in the cortical cells (Figures 5F and 5I), whereas those ofCDPK7 andCDPK10were highest in the outer part of rice roots at36 h under stagnant conditions (Figures 5Gand5H). These resultsindicate that CDPK5 and CDPK13 expression is most closelyassociated with aerenchyma formation.

Direct Activation of Os-RBOHH by Os-CDPKs inN. benthamiana Leaves

To examine the activation of the RBOHH-mediated ROS pro-duction by CDPKs, Os-RBOHH and Os-CDPKs were coex-pressed in N. benthamiana leaves, and subsequent ROSproduction was quantified by a photon-counting system. Forthese experiments, we used three forms of CDPK: the full-length(wild-type) form, the constitutively active VK form (Harper et al.,1994), and theK/M form (Kapiloff et al., 1991),which is defective inprotein kinase activity (Figure 6A).Coexpression of the FLAG-tagged RBOHH with the hemag-

glutinin (HA)-tagged CDPK5VK or CDPK13VK strongly inducedROS production inN. benthamiana leaves, whereas the inductionby HA-tagged CDPK7VK or CDPK10VK was relatively weak(Figure 6B). By contrast, coexpression of FLAG-tagged GUS,which was used as a negative control, with the VK forms of theHA-tagged CDPKs did not significantly induce ROS production(Figure 6B). The amount of RBOHH (which was detected using ananti-FLAG antibody) in the microsomal fraction was lower whenRBOHH was coexpressed with CDPK10VK or CDPK13VK thanwhen itwascoexpressedwithCDPK5VKorCDPK7VK (Figure6C).The protein levels of CDPKVKs except for CDPK7VK (which wasdetectedusingananti-HAantibody)werecomparable (Figure6C).Coexpression of RBOHHwith CDPK5WT or CDPK13WT inducedROS production weakly, and the strong ROS production inducedby coexpression of RBOHH with CDPK5VK or CDPK13VK wassuppressed by the K/M mutation (Figure 6D). The protein level ofRBOHHwas relatively lowerwhenRBOHHwas coexpressedwithCDPK13VK than when it was coexpressed with CDPK5VK, andthe protein level of CDPK13VK was lower than that of CDPK5VK(Figure 6E). These results indicate that CDPK5 and CDPK13 (butnotCDPK7andCDPK10) strongly induceRBOHH-mediatedROSproduction.

Ethylene-Dependent ROS Production duringAerenchyma Formation

To examine the effect of ethylene on the RBOH-mediated ROSproduction during inducible aerenchyma formation in rice roots,10-d-old aerobically grown rice seedlings were treated with theethylene perception inhibitor 1-methylcyclopropene (1-MCP; 2 ppm)

Figure 2. Expression of RBOH Genes in Rice Roots.

(A) Absolute transcript levels of RBOH genes at 10 mm (6 2 mm) from thetips of adventitious roots of rice seedlings grownunder aerated or stagnantconditions for 36 h.(B)and (C)Time-course relative transcript levelsofRBOHA (B)andRBOHH(C) at 10 mm (62 mm) from the root tips under aerated or stagnantconditions.(D) Isolation of the central cylinder (CC), cortex (Co), and outer part of root(OPR) from theparaffin-embeddedsections of rice roots at 10mm (62mm)using laser microdissection (LM). The 20-d-old aerobically grown riceseedlings were subjected to the aerated or stagnant treatments for 36 h.Bars = 100 mm.(E) and (F) Relative transcript levels of RBOHA (E) and RBOHH (F) in thecentral cylinder, cortex, and outer part of root at 10 mm (62 mm) from theroot tips under aerated or stagnant conditions.

The 20- to 40-mm roots ([A] to [C]) and 30- to 50-mm roots ([E] and [F]) ofthe 10-d-old and 20-d-old aerobically grown rice seedlings, respectively,were subjected to the treatments. The gene encoding transcription initi-ation factor IIE, TFIIE, was used as a control. Values aremeans6 SD (n = 3).Significant differences between aerated and stagnant conditions at**P < 0.01 and *P < 0.05 (two-sample t test).

778 The Plant Cell

under stagnant conditions. 1-MCP suppressed the induction ofaerenchyma formationat10mmfrom the tipsof adventitious rootsat48 h under stagnant conditions (Figure 7A). It also suppressed theincreased H2O2 content in the apical regions (at 5–25 mm) of ad-ventitious rootsat36h (Figure7B).Theexpression levelofALCOHOLDEHYDROGENASE2 (ADH2), which functions in ethanol fermenta-tion, is stimulated by ethylene (Fukao et al., 2006). At 36 h, 1-MCPalso suppressed the inductions of ADH2 and RBOHH expression,

but not CDPK5 and CDPK13 expression, at 10 mm (62 mm) fromthe tips of adventitious roots (Figures 7C to 7F). These resultsindicate that RBOHH expression, but not CDPK5 and CDPK13expression, is regulated through ethylene signaling.

Phenotype of the RBOHH-Targeted CRISPR/Cas9Transgenic Lines

Six independent RBOHH-CRISPR/Cas9 (RHC) T0 plant lines(RHC#1 to #6) were regenerated from transformed rice (cv Nip-ponbare) calli (Supplemental Figure 6A). Sequence analysis ofgenomic DNA obtained from leaves of the regenerated plantsrevealed that all plant lineshad insertionsordeletions that resultedin frameshifts and premature stop codons: three of the RHC lines(#2, #3, and #4) were homozygously mutagenized, and the otherthree lines (#1, #5, and #6) retained the wild-type RBOHH se-quence (Supplemental Figure 6B).Subsequently, four RHC lines (#2, #3, #4, and #6) were transferred

to aerated nutrient solution and grown for 20 d. The RHC lines with20-d-old aerobically grownbackgroundwild typewere transferred toaerated or stagnant conditions. At 48 h under stagnant conditions,aerenchyma formation in adventitious roots of the vector control (VC)and RHC#6 lines was not significantly different from that of the wildtype, whereas aerenchyma formation in the homozygously muta-genized RHC#2, #3, and #4 lines was significantly reduced (Figures8Aand8B;Supplemental Figure7). Thehomozygouslymutagenizedgenotypes of RHC#2, #3, and #4 lines, and the wild type and mu-tagenized RBOHH sequences of the RHC#6 line were confirmed bysequence analysis of mRNA from the seedling roots (SupplementalFigure6C). TheH2O2content in theapical regions (at 5–25mm)of theadventitious rootswas reduced in theRHC#2,#3, and#4 linesat36hunder stagnant conditions, whereas the H2O2 content in the RHC#6line was comparable with the contents in the wild type and VC line(Figure8C).Asignificant reductionofaerenchymaformationwasalsodetected in the RHC#2, #3, and #4 lines grown under stagnantconditions for 1 week (Supplemental Figure 8).RBOH expression is considered a reliable indicator of ROS pro-

duction (Desikanetal., 1998;Linetal.,2009). Indeed, in rice roots,DPIand EGTA, both of which suppressed the increased H2O2 contentunder stagnant conditions (Figures 4B and 4D), also suppressed theinduction of RBOHH expression (Supplemental Figure 9). In theRHC#2, #3, and#4 lines,RBOHHexpressionat 10mm (62mm)wassignificantly reducedwhencomparedwith thewild typeaswell as theVC and RHC#6 lines at 36 h under stagnant conditions (Figure 8D).These results further support the idea that the reduced aerenchymaformation in the RHC lines under stagnant conditions is caused bya defect in RBOH-mediated ROSproduction. It should be noted thatthree homozygously mutagenized RHC lines (#2, #3, and #4) werefound to be completely sterile (Supplemental Table 1).

DISCUSSION

NADPH Oxidase RBOHH-Mediated ROS Production IsEssential for Ethylene-Dependent Aerenchyma Formation

In this study, we found that the expression of the gene encod-ing the NADPH oxidase RBOHH was closely associated with

Figure 3. Accumulation of ROS in Rice Roots.

(A) Time course of H2O2 content at 5 to 25mm from the tips of adventitiousroots of rice seedlings grown under aerated or stagnant conditions. H2O2

content was quantified as the H2O2 (pmol) per gram fresh weight (gFW) ofroot segments. Values are means 6 SD (n = 6).(B)Crosssectionsat10mmfromthe tipsofNBT-stainedadventitious rootsof rice seedlings grown under aerated or stagnant conditions for 36 h.Collapsed cells and NBT-stained cells are indicated by red or blue ar-rowheads, respectively. Bars = 50 mm.(C) and (D) Numbers of collapsed cells (C) and NBT-stained cells (D) ineachcortical cell layerat10mmfromthe root tipsunderaeratedorstagnantconditions for 36 h. Values are means 6 SE (n = 12).The 20- to 40-mm roots of the 10-d-old aerobically grown rice seedlingswere subjected to the treatments. Significant differences between aeratedand stagnant conditions at **P < 0.01 and *P < 0.05 (two-sample t test).

RBOHH Regulates Root Aerenchyma Formation 779

aerenchyma formation in adventitious roots of rice under stag-nant conditions (Figures 1B, 2A, 2C, and 2F). The ethylene ac-cumulation in rice roots was significantly increased at 24 h(Supplemental Figure 2C), in agreement with our previous studies(Yamauchi et al., 2015, 2016). RBOHH expression was stronglyinducedby transfer to stagnant conditions, peaking at 36 h (Figure2C). The increase in H2O2 was partly suppressed by the ethyleneperception inhibitor 1-MCP (Figure7B). These resultssuggest thatRBOHH-mediated ROS production is stimulated by ethyleneunder stagnant conditions. In rice roots, 1-MCP suppressedboth aerenchyma formation and RBOHH expression (Figures 7Aand 7D), similar to its effects on aerenchyma formation andZm-RBOHH expression inmaize primary roots (Rajhi et al., 2011).

Aerenchyma formation in rice roots was reduced by DPI (Figure4A). Moreover, the H2O2 level and aerenchyma formation weresuppressed by the disruption of RBOHH function (Figures 8B and8C; Supplemental Figures 7 and 8). These results further supportthe idea that RBOHH-mediated ROS production is essential foraerenchyma formation in rice roots. DPI suppresses aerenchymaformation induced by waterlogging in maize primary roots(Yamauchi et al., 2011) and suppresses aerenchyma formationinduced by the ethylene precursor ACC in seminal roots of wheat(Yamauchi et al., 2014). Application of H2O2 or the ethylene-releasing compound ethephon promotes lysigenous aeren-chyma formation in the internodes of rice (Steffens et al., 2011).Taken together, these findings indicate that control of lysigenousaerenchyma formation by ethylene-induced RBOH-mediatedROS production is a common feature of gramineous plants.

Functional Conservation and Diversity of Os-RBOHH

Os-RBOHH and Zm-RBOHH belong to the same clade asOs-RBOHB (i.e., clade I in Supplemental Figure 3). Os-RBOHBand Os-RBOHH were identified as candidate defense-relatedproteins in the plasma membrane microdomains (which locali-zation is essential to their function) of rice suspension cells thatwere inoculated with rice blast fungus (Magnaporthe oryzae)(Nagano et al., 2016). Knockdown of Os-RBOHB resulted ina greater vulnerability to rice blast fungus, whereas knockdownof Os-RBOHH did not increase the response to chitin elicitortreatment (whichmimics fungus inoculation) (Naganoet al., 2016).Os-RBOHH and Zm-RBOHH, but not Os-RBOHB, belong to thesame subclade in clade I of the RBOH tree (Supplemental Figure3); thus, Os-RBOHH and Zm-RBOHHmay have specialized rolesin inducible aerenchyma formation in rice and maize roots, re-spectively. Two other gramineous plants, sorghum (Sorghumbicolor) and foxtail millet (Setalia italica), both of which formlysigenous aerenchyma in the roots (McDonald et al., 2002;Matsuura et al., 2016), have closely related homologs toOs-RBOHH, Sb-RBOH (accession number XM_002442219),and Si-RBOH (accession number XM_004962769). It would beinteresting to investigate whether these RBOHs are also involvedin ROS-mediated aerenchyma formation.Under anoxic (anaerobic) conditions, a strong but transient

increase of H2O2 accumulation was detected in Arabidopsisseedlings (Pucciariello et al., 2012). In an Arabidopsis mutant ofhypoxia-responsive universal stress protein 1, which interactswith the At-RBOHD complex, the H2O2 increase was reduced(Gonzali et al., 2015). By contrast, the transcript level of At-ADH1was not affected by the lack of functional At-RBOHD underanoxic conditions (Pucciariello et al., 2012). At-RBOHB belongsto the same clade as Os-RBOHB and Os-RBOHH (SupplementalFigure 3). At-RBOHBmRNA accumulation in polysomes, whichare active in protein synthesis, is significantly increased underhypoxic (oxygen-deficient) conditions (Branco-Price et al.,2005). Under the same conditions, the elevated H2O2 level aswell as the increased transcript level of At-ADH1 is suppressedby DPI (Baxter-Burrell et al., 2002). Os-RBOHB andOs-RBOHHexpression in roots is significantly induced under stagnantconditions (Figure 2A). These results suggest that one ofthe conserved functions of clade I RBOHs (e.g., At-RBOHB,

Figure 4. Effects of DPI and EGTA on Aerenchyma Formation and H2O2

Accumulation in Rice Roots.

(A) The percentage of aerenchyma of root cross-sectional area at 10 mmfrom the tips of adventitious roots of rice grown under aerated conditions,or stagnant conditions with 0, 0.05, or 0.25 mMDPI treatment for 48 h. Verylittle aerenchyma formed under aerated conditions.(B) The H2O2 content in rice roots at 5 to 25 mm from the root tips underaerated conditions, or stagnant conditions with or without 0.25 mM DPItreatment for 36 h.(C) The percentage of aerenchyma of root cross-sectional area at 10 mmfrom the root tips under aerated conditions, or stagnant conditions with 0,50,or100mMEGTAtreatment for 48h.Very little aerenchyma formedunderaerated conditions.(D) The H2O2 contents in rice roots at 5 to 25 mm from the root tips underaerated conditions, or stagnant conditions with or without 100 mM EGTAtreatment for 36 h.The 20- to 40-mm roots of the 10-d-old aerobically grown rice seedlingswere subjected to the treatments. Values are means 6 SD (n = 9 [A] and[C], and n = 6 [B] and [D]). Different lowercase letters denote significantdifferences among different conditions (P < 0.05, one-way ANOVA andthen Tukey’s test for multiple comparisons).

780 The Plant Cell

Os-RBOHB, and Os-RBOHH) is to transduce the signal underoxygen-deficient conditions. Since Arabidopsis does not formlysigenous aerenchyma, rice (and other gramineous plants)appears to have acquired a role for RBOH in aerenchyma for-mation to increase fitness under oxygen-deficient conditionsassociated with soil waterlogging. During evolution, Os-RBOHHand the other gramineous RBOHs in the same subclade appearto have become specialized as signal transducers for aeren-chyma formation. Some plant RBOHs mediate multiple bio-logical functions (Suzuki et al., 2011). TheRBOHH knockout lineswere found to be completely sterile (Supplemental Table 1),suggesting that RBOHH-mediated ROS production is also es-sential for reproduction in rice plants.

Activation of RBOHH-Mediated ROS Production throughCa2+ Signaling Is Involved in Aerenchyma Formation

ROS production during inducible aerenchyma formation wassuppressed by an inhibitor of cytosolic Ca2+ influx from theapoplast (Figure 4D). In maize suspension-cultured cells underanoxic conditions, mitochondria are the main source of cytosolicCa2+ influx (Subbaiah et al., 1998). In this study, aerenchymaformation was partly suppressed by 10 mM ruthenium red(Supplemental Figure 4). High concentrations (10–25 mM) of ru-thenium red inhibit cytosolic Ca2+ influx from the apoplast(Subbaiah et al., 1994), suggesting that the effect of rutheniumred in rice roots is due to the inhibition of cytosolic Ca2+ influx fromthe apoplast. Indeed, aerenchyma formation in maize roots wasalso blocked by EGTA and by a high concentration (25 mM) ofruthenium red (He et al., 1996). Together, these findings suggestthat apoplastic Ca2+ is a major source of the cytosolic Ca2+ influxcontributing to aerenchyma formation in roots of gramineousplants.Under stagnant conditions, the expression of CDPK5

and CDPK13 as well as RBOHH was greater in the corticalcells than in the cells of other root tissues (Figures 2F, 5F,and 5I). In addition, CDPK5 and CDPK13 strongly inducedRBOHH-mediated ROS production in N. benthamiana leaves(Figure 6B). Since RBOHH expression was positively autor-egulated through RBOH-mediated ROS signaling (Figure 8D;Supplemental Figure 9A), CDPK5- and/or CDPK13-mediatedactivation of RBOHH might autoactivate RBOHH expression.

Figure 5. Expression of CDPK Genes in Rice Roots.

(A) Absolute transcript levels of CDPK genes at 10 mm (62 mm) from thetips of adventitious roots of rice seedlings grownunder aerated or stagnantconditions for 36 h.

(B) to (E) Time-course relative transcript levels of CDPK5 (B), CDPK7 (C),CDPK10 (D), and CDPK13 (E) at 10 mm (62 mm) from the root tips underaerated or stagnant conditions.(F) to (I) Relative transcript levels of CDPK5 (F), CDPK7 (G), CDPK10 (H),andCDPK13 (I) in thecentral cylinder (CC), cortex (Co), andouterpart of theroot (OPR) at 10 mm (62 mm) from the root tips under aerated or stagnantconditions for 36 h. The 20-d-old aerobically grown rice seedlings weresubjected to the treatments.The 20- to 40-mm roots ([A] to [E]) and 30- to 50-mm roots ([F] to [I]) of the10-d-old and 20-d-old aerobically grown rice seedlings, respectively, weresubjected to the treatments. The gene encoding transcription initiationfactor IIE, TFIIE, was used as a control. Values are means 6 SD (n = 3).Significant differences between aerated and stagnant conditions at**P < 0.01 and *P < 0.05 (two-sample t test).

RBOHH Regulates Root Aerenchyma Formation 781

This would create a positive feedback circuit resulting inlong-lasting RBOHH-mediated ROS production. In addition,EGTA, which has been shown to suppress CPK-dependentAt-RBOHD phosphorylation (Kadota et al., 2014), suppressedinduction of Os-RBOHH expression under stagnant conditions(Supplemental Figure 9B). This result supports the idea thatCDPK-mediated ROS production by RBOHH autoactivatesRBOHH expression. However, EGTA may also weaken the ac-tivity of RBOH protein by preventing Ca2+ from binding toEF-hand motifs (helix-loop-helix motifs found in many calcium

binding proteins) in the N-terminal region of RBOHH. Suchbinding has been shown to stimulate the activity of RBOHs(Kimura et al., 2012). The supply of newly synthesizedNb-RBOHB followed by CDPK activation in response to plantpathogen-derived components seems to permit a sustained andmassive ROS production in N. benthamiana (Adachi et al., 2015).Taken together, these findings suggest that the massive pro-duction of ROS during aerenchyma formation in rice roots ismediated by autoactivation of RBOHH by CDPK5 and/orCDPK13.

Figure 6. ROS Production Mediated by Os-RBOHH and Os-CDPKs in N. benthamiana Leaves.

(A) Structures of CDPKwith variable (V), kinase (K), junction, and calmodulin-like domains. The wild type (WT) includes all domains of the CDPK. The VK isa truncated variant that lacks the junction and calmodulin-like domains. The K/M is a derivative of the VKwith substitution of the Lys (K) of the ATP bindingmotif to Met (M). HA indicates HA tag. N and C indicate the N terminus and C terminus of CDPK protein, respectively.(B) ROS production in N. benthamiana leaves in which the C-terminal HA-tagged Os-CDPK5VK-HA, Os-CDPK7VK-HA, Os-CDPK10VK-HA, orOs-CDPK13VK-HA protein was coexpressed with the N-terminal FLAG-tagged FLAG-GUS or FLAG-Os-RBOHH protein. ROS production wasmeasuredby chemiluminescence mediated by L-012 at 2 d after infiltration of Agrobacterium into the leaves. White circles indicate areas infiltrated with L-012.(C) Immunoblot analyses using anti-FLAG or anti-HA antibody. Microsomal proteins were prepared from the leaves shown in (B). Protein loads weremonitored by Coomassie Brilliant Blue (CBB) staining of the bands corresponding to the Rubisco large subunit (RBCL). Blue, orange, and magentapentagrams indicate the signals of GUS, Os-RBOHH, and Os-CDPK variants.(D)ROS production inN. benthamiana leaves in which the GUS-HA protein or the wild-type, VK, or K/M form of Os-CDPK5-HA or Os-CDPK13-HA proteinwas coexpressed with the FLAG-Os-RBOHH protein. ROS production was measured as described in (B).(E) Immunoblot analyses using anti-FLAG or anti-HA antibody. Microsomal proteins were prepared from the leaves shown in (D). Protein loadswere monitored by Coomassie Brilliant Blue staining of the bands corresponding to the Rubisco large subunit. Blue, orange, and magenta pentagramswere as described in (C).Valuesaremeans6SE (n=10 [B]andn=9 [D]).Different lowercase lettersdenotesignificantdifferencesamongdifferentconditions;P<0.15 (B)andP<0.05(D), one-way ANOVA and then Tukey’s test for multiple comparisons.

782 The Plant Cell

Target Specificities of Group I CDPKs in Vivo

In rice roots, the expression of CDPK5 and CDPK13 (Figures 5Fand 5I), but not CDPK7 and CDPK10 (Figures 5G and 5H), wassignificantly induced in the cortex, where strong induction ofaerenchyma formation and RBOHH expression were both de-tected (Figures 1A and 2F). Moreover, transcript levels of CDPK5and CDPK13 were highest in the cortex (Figures 5F and 5I),whereas transcript levels of CDPK7 and CDPK10 were highest inthe outer part of the roots (Figures 5G and 5H). These resultssuggest that CDPK5 and CDPK13 have more essential roles inregulating RBOHH activity during aerenchyma formation thanother group I CDPKs, such as CDPK7 and CDPK10. However, asinduction of expression ofCDPK5 (Figure 5B)was earlier than thatofCDPK13 (Figure 5E),CDPK5andCDPK13might phosphorylateRBOHH in a stepwise manner and thus might not be redundant.Os-CDPK5 and Os-CDPK13 strongly induced Os-RBOHH-

mediated ROS production in N. benthamiana leaves (Figure 6B).Ca2+-activated CDPK-mediated ROS production has a role inimmune responses in plants (Kobayashi et al., 2007; Asai et al.,2013; Dubiella et al., 2013; Kadota et al., 2014), with many group ICDPKs (At-CPK4,At-CPK5,At-CPK6,At-CPK11,St-CDPK4, andSt-CDPK5) (Supplemental Figure 5), directly phosphorylatingRBOHs. Double (cpk5 cpk6), triple (cpk5 cpk6 cpk11), and qua-druple (cpk5 cpk6 cpk11 cpk4) mutants showed progressivelyreducedROSproduction induced by the bacterial elicitor flagellin,indicating that these At-CPKs coordinately regulate ROS pro-duction during the immune response in Arabidopsis (Boudsocqet al., 2010). The rice homologs Os-CDPK5 and Os-CDPK13, butnot Os-CDPK7 andOs-CDPK10, belong to the same subclade asAt-CPK5, At-CPK6, St-CDPK4, and St-CDPK5 (SupplementalFigure 5), which directly phosphorylate At-RBOHD (Dubiella et al.,2013; Kadota et al., 2014) andSt-RBOHB (Kobayashi et al., 2007),respectively. By contrast, At-CPK4 and At-CPK11, both of whichalso directly phosphorylate At-RBOHD, do not belong to thesame subclade as At-CPK5 and At-CPK6 (Supplemental Figure 5).As Os-CDPK7 and Os-CDPK10 did not significantly inducethe Os-RBOHH-mediated ROS production in N. benthamianaleaves, Os-CDPK7 and/or Os-CDPK10 might have substratesother than Os-RBOHH.Having theproper subcellular localization isessential forCDPKs

to phosphorylate their target proteins in vivo (Latz et al., 2013;Simeunovic et al., 2016). Swapping experiments of regions foreach domain between two different CDPKs showed that thevariable domains confer the substrate specificities of CDPKsinvivobydictating their proper subcellular localizations (Asai et al.,2013). The lower ROS production level upon coexpression ofOs-CDPK7 and Os-RBOHH in N. benthamiana leaves (Figure 6B)might be caused by the lower protein level of Os-CDPK7 in themicrosomal fraction (Figure 6C). Therefore, even though thetranscript level ofCDPK7was lower in the cortex than in the outerpart of the rice roots (Figure 5G), we cannot rule out the possibilitythat CDPK7 also partly contributes to RBOHH activation duringaerenchyma formation. Further studies are needed to identify thesubcellular localizations of the CDPKs used in this study to clarifytheir substrate specificities.Direct activationofRBOHsbyCDPKshasa role inplant immune

responses (Kobayashi et al., 2007; Asai et al., 2013; Dubiella et al.,

Figure7. Effectof1-MCPonAerenchymaFormation,H2O2Accumulation,and Gene Expression in Rice Roots.

(A) The percentage of aerenchyma of root cross-sectional area at10 mm from the tips of adventitious roots of rice grown under aeratedconditions, or under stagnant conditions with or without 2 ppm 1-MCPtreatment for 48 h. Very little aerenchyma formed under aeratedconditions.(B) The H2O2 content in rice roots at 5 to 25 mm from the root tips underaerated conditions, or under stagnant conditions with or without 2 ppm1-MCP treatment for 36 h.(C) to (F)Relative transcript levels ofADH2 (C),RBOHH (D),CDPK5 (E),and CDPK13 (F) at 10 mm (62 mm) from the root tips under aeratedconditions, or under stagnant conditions with or without 2 ppm 1-MCPtreatment for 36 h. The gene encoding transcription initiation factor IIE,TFIIE, was used as a controlThe 20- to 40-mm roots of the 10-d-old aerobically grown riceseedlings were subjected to the treatments. Values are means 6

SD (n = 9 [A], n = 6 [B], and n = 3 [C] to [F]). Different lowercaseletters denote significant differences among different conditions(P < 0.05, one-way ANOVA and then Tukey’s test for multiplecomparisons).

RBOHH Regulates Root Aerenchyma Formation 783

2013; Kadota et al., 2014). Our results suggest that CDPK5 and/orCDPK13 directly activated RBOHH in rice roots during aeren-chyma formation (Figure 6). RBOH-mediatedROSproduction andcytosolic freeCa2+elevationhavebeen reported tocontrol diverse

biological processes in plants (Mittler et al., 2011; Marino et al.,2012). Our results suggest that activation of RBOHs by CDPKsunderliesmany of these processes. Further studies on the variouscombinations of CDPKs and RBOHs in diverse biological pro-cesses will be required to better understand the in vivo targetspecificities of group I CDPKs.

Mechanisms Underlying Cortical Cell-SpecificAerenchyma Formation

Lysigenous aerenchyma formation was confined to the corticalcells in rice roots (Figures 1A and 3C). Root cortical cells formingaerenchyma are thought to be sensitive to ethylene (Justin andArmstrong, 1991). Therefore, sensitivity to ethylenemight underliethe initiation of PCD during inducible aerenchyma formation. In-deed, RBOHH expression, which was induced by ethylene understagnant conditions (Figure 7D), was greater in the cortical cellsthan in the cells of other tissues (Figure 2F). CDPK5 and CDPK13expression was also greater in the cortical cells under stagnantconditions (Figures 5F and 5I). Therefore, ethylene-inducedRBOHH expression and CDPK5- and/or CDPK13-mediated

Figure 8. Aerenchyma Formation, H2O2 Accumulation, and RBOHH Ex-pression in Roots of RBOHH-Targeted CRISPR/Cas9 Transgenic Lines.

(A) Cross sections at 20 mm from the tips of adventitious roots of the wildtype (WT), VC, andRBOHH-targetedCRISPR/Cas9 (RHC) transgenic lines(RHC#2, #3, #4, and #6) grown under aerated or stagnant conditions for48 h. Lysigenous aerenchyma is indicated by arrowheads. Bars = 100 mm.(B) The percentage of aerenchyma of root cross-sectional area at 20 mmfrom the root tips of the wild type, VC, and RHC lines under aerated orstagnant conditions for 48 h.(C) The H2O2 content at 5 to 25 mm from the root tips of the wild type, VC,and RHC lines under aerated or stagnant conditions for 36 h.(D) Expression of RBOHH at 10 mm (62 mm) from the root tips of the wildtype, VC, and RHC lines under aerated or stagnant conditions for 36 h.RHC#2, #3, and #4 are homozygously mutagenized lines, and RHC#6retains the wild-type RBOHH gene in the genome. The lengths of rootsimmediately before the treatments (at 0 h) were 30 to 50 mm. Values aremeans 6 SD (n = 3). Significant differences between the wild type and thetransgenic lines under aerated or stagnant conditions at **P < 0.01 and*P < 0.05 (two-sample t test).

Figure 9. Model for Inducible Aerenchyma Formation in Rice Roots.

During inducible aerenchyma formation in rice roots under waterloggedconditions, ethylene biosynthesis is increased through the induction ofACS1 and ACO5 expression. The low rate of gas diffusion under water-logged conditions also contributes to enhance ethylene accumulation inrice roots. In root cortical cells, the elevated ethylene enhances the ex-pression of RBOHH. Cytosolic Ca2+ influx from apoplast is stimulated,resulting in RBOHH protein activation by CDPK5 and/or CDPK13, theexpression of which is induced under oxygen-deficient conditions. Theapoplastic O2

$2 is spontaneously or enzymatically converted to H2O2, andthe H2O2 diffuses or is transported into the cytosol through the plasmamembrane. Thus, the increased apoplastic O2

$2 production mediated byRBOHH leads to the increasing H2O2 level in the apoplast and/or cytosol.Finally, the increased amounts of apoplastic and/or intracellular ROS (O2

$2and/or H2O2) trigger the induction of PCD and subsequent lysigenousaerenchyma formation in the cortical cells of rice roots.

784 The Plant Cell

activation of RBOHH protein are essential for strong ROS pro-duction in root cortical cells.

The cell collapse during constitutive aerenchyma formation inrice roots starts from the mid-cortex (Kawai et al., 1998). Duringinducible aerenchyma formation in rice roots under stagnantconditions, the collapsed cells and O2

$2 accumulating cells weremost frequently detected in the mid-cortex (Figures 3B to 3D).Moreover, the collapsed cells and O2

$2 accumulation expandedradially from themid-cortical cells to the adjacent cells (Figure 3B).Together, thesefindings suggest that the signal for triggeringPCDis independently regulated in each of the cortical cells. It wasproposed that the sensitivity of initiator cells (i.e., mid-corticalcells) to ethylene is higher than that of other cortical cells (Visserand Bögemann, 2006). According to this hypothesis, RBOHHexpression may be strongly induced in the mid-cortical cells.RBOH-mediated ROS production is involved in cell-to-cellcommunication (Mittler et al., 2011). During the systemic signalpropagation in Arabidopsis, RBOH-mediated apoplastic O2

$2

production stimulatesRBOHactivity of the adjacent cells, and thiscontinuous activation of RBOH is essential for the systemic signaltransduction during responses to external stimuli (Miller et al.,2009; Dubiella et al., 2013). Further studies are needed to un-derstandcell-to-cell signal spreadingmechanisms triggeringPCDduring aerenchyma formation in rice roots.

Model for Molecular Mechanism UnderlyingAerenchyma Formation

Based on the preceding results, we propose amodel for RBOHH-mediated ROS production during ethylene-induced aerenchymaformation in rice roots (Figure 9). During aerenchyma formation inwaterlogged rice roots, the amount of ethylene is increased byenhancement of ethylene biosynthesis and by the low diffusionrate of ethylene. Ethylene then stimulates RBOHH expression.RBOHH protein is subsequently activated by CDPK5 and/orCDPK13 to increase apoplastic O2

$2 production. Apoplastic O2$2

is spontaneously or enzymatically converted to H2O2, which dif-fuses or is transported into the cytosol through the plasmamembrane. Finally, the increased amounts of apoplastic and/orintracellular ROS (O2

$2 and/or H2O2) lead to the induction of PCD(i.e., lysigenous aerenchyma formation) in the cortical cells of riceroots. Further studies are needed to see if other factors are in-volved in this type of PCD.

METHODS

Plant Materials and Growth Conditions

Rice (Oryza sativa cv Shiokari or cv Nipponbare) seeds were surfacesterilized in 0.5% (v/v) sodium hypochlorite for 30 min and rinsed thor-oughly with deionized water. The seeds were germinated on Petri disheswith deionized water in a growth chamber at 28°C under dark conditions.After 2 d, germinated seeds were placed on a meshfloat with an aeratedquarter strength nutrient solution at 28°C under constant light conditions(photosynthetically active radiation, 200–250 mmol m22 s21) for 4 d.Composition of the full-strength nutrient solution is described by Colmeret al. (2006). Six-day-old seedlings were transferred to 5-liter pots (8–12plants per pot, 250 mm height 3 120 mm length 3 180 mm width) con-taining an aerated full-strength nutrient solution (aerated conditions) or

stagnant solution (stagnant conditions). Stagnant solution contained0.1%(w/v) dissolved agar andwasdeoxygenated (dissolvedO2, <0.5mg/L) priorto use by flushing with N2 gas (Wiengweera et al., 1997).

Nicotiana benthamiana plants were grown at 23°C under a 16-h pho-toperiod and an 8-h dark period in environmentally controlled growthcabinets.

Anatomical Observations of Roots

Root cross sections were prepared from 4-mm-long segments excisedfrom adventitious roots of rice seedlings. Segmentswere prepared at eachposition of adventitious roots. Cross sections were prepared by handsectioning with a razor blade. Each section was photographed using anopticalmicroscope (BX60;Olympus)with aCCDcamera (DP70;Olympus).Outlines of aerenchyma in each cross section were traced with a cursor,and their areas were quantified with ImageJ (version 1.43u; NationalInstitutes of Health) to obtain the percentage of each cross section oc-cupied by aerenchyma. Three independent experiments, each with threereplications, were performed. Values are expressed as the average of thenine measurements.

RT-qPCR Analysis

For each growth condition, 4-mm segments were cut from adventitiousroots (from 8–12 mm from the root tips). Segments from three roots fromone, two, or three plants were pooled and ground in liquid nitrogen. TotalRNA from the ground tissues was extracted using an RNeasy plant mini kit(Qiagen) according to the manufacturer’s instructions. For RT-qPCR, 1 ngtotal RNA was used as template. Transcript levels were measured usinga StepOnePlus real-time PCR system (Applied Biosystems) and One StepSYBRPrimeScriptRT-PCRKit II (TakaraBio). APCR fragmentofeachgenewaspurifiedandquantified, and thenwasused to drawstandard curves forabsolute quantification. Transcript levels were normalized to the level ofTFIIE (transcription initiation factor IIE), which was not largely affected bydifferent growth conditions or by different chemical treatments. Two orthree independent experiments were performed, and the results of eachexperimentwere comparable.Datawereexpressedas theaverageof threereplications. Primer sequences used for the RT-qPCR are shown inSupplemental Table 2.

Phylogenetic Analysis

The amino acid sequences for C-terminal domains that include the ferricreductase-like transmembrane component, flavin adenine dinucleotidebinding, and ferric reductase nicotinamide adenine dinucleotide bindingdomains of RBOHs (Supplemental File 1) and protein kinase domain ofCDPKs (Supplemental File 2) of Arabidopsis thaliana and rice wereobtained from The Arabidopsis Information Resource (https://www.arabidopsis.org) and Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/) or MSU Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml) databases, respectively. The aminoacid sequences of RBOHs andCDPKs of other plant species were obtainedfrom National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov). The neighbor-joining phylogenetic trees were constructed as de-scribed by Yamauchi et al. (2016).

Ethylene Measurement

For each condition (aerated and stagnant), three independent experimentswere performed. In each experiment, the underground parts were excisedfrom 20-d-old rice seedlings (five to eight plants for each sample) andwereplaced in a container with saturated sodium chloride solution. The gasreleased from the rice seedlings under vacuumconditionswas collected in

RBOHH Regulates Root Aerenchyma Formation 785

a test tube using a funnel, transferred to a gas chromatography vial, andmeasured by gas chromatography (GC353; GL Sciences). One mea-surement was made for two of the experiments and two measurementswere made for one of the experiments, for a total of four measurements.Values were expressed as an average of the four measurements.

Laser Microdissection

For each growth condition, 4-mm segments were cut from adventitiousroots (from 8–12 mm from the root tips). Segments were fixed in 100%methanol. After fixation, the samples were embedded in paraffin andsectioned at a thickness of 16 mm. Serial sections were placed onto PENmembrane glass slides (Life Technologies) for laser microdissection asdescribed byTakahashi et al. (2010). The central cylinder, cortex, andouterpart of roots were collected from the tissue sections using a VeritasLCC1704 laser microdissection system (Life Technologies). The tissuesections of two or three roots were pooled, and total RNA from three poolsof the tissue sectionswasextracted usingaPicoPureRNA isolation kit (LifeTechnologies) according to themanufacturer’s instructions. The extractedtotal RNA was quantified with a Quant-iT RiboGreen RNA reagent and kit(Life Technologies) according to the manufacturer’s instructions. Thequality of total RNAwas evaluated using a RNA 6000 Pico kit on an Agilent2100 Bioanalyzer (Agilent Technologies).

H2O2 Quantification

For each growth condition, 20-mm segments were cut from adventitiousroots (from 5–25mm from the root tips). Segments from two or three rootsfromoneor twoplantswerepooledandground in liquid nitrogen.H2O2wasextracted from the ground tissues and quantified with an Amplex Redhydrogen peroxide/peroxidase assay kit (Thermo Fisher Scientific) ac-cording to the manufacturer’s recommendations. For each condition, twomeasurements were made for each of three independent experiments.Values were expressed as an average of the six measurements.

NBT Staining

The histochemical detection of O2$2 by NBT staining was performed as

described by Hernández et al. (2001) with minor modifications. Rootsegments were infiltrated under vacuum for 1 h at room temperature with1mg/mLNBT (WakoChemical Industries) in 25mMHEPES buffer (pH7.6).After the infiltration, crosssectionsofone, two, or three root segments fromeach of six to eight plants were prepared and photographed as describedabove. Subsequently, the numbers of collapsed cells and NBT-stainedcells in each cortical cell layer were counted. Values were expressed as anaverage of 12 replications. Two identical experiments were subsequentlyperformed with comparable results.

Chemical Treatments

For the DPI, EGTA, and ruthenium red treatments, rice seedlings weretransferred to 2-liter pots (four plants per pot, 250 mm height 3 80 mmlength 3 120 mm width) including nutrient solution with appropriateconcentrations of each chemical; 500 mM DPI (Sigma-Aldrich) was addedto prepare the nutrient solutions with 0.05 or 0.25 mM DPI; 300 mM EGTA(Dojindo Molecular Technologies) was added to prepare the nutrientsolutionswith 50 or 100 mMEGTA; 100mM ruthenium red (WakoChemicalIndustries) was added to prepare the nutrient solutions with 10 or 25 mMruthenium red. Stock solutions of DPI and ruthenium red were dissolved indeionized water and that of EGTA was dissolved in 1 N KOH.

For the 1-MCP treatment, rice seedlings were transferred to 2-liter potswith nutrient solution, and then the pots were placed in a tightly closedcontainer with or without 2 ppm 1-MCP. The 1-MCP was renewed at 24 h

after the start of the treatment. The concentrationof 1-MCPwascalculatedas described by Visser and Bögemann (2006).

Agrobacterium tumefaciens-Mediated Transient Expression andROS Measurement in N. benthamiana Leaves

ThecDNA fragmentsof full-lengthRBOHH,CDPK5, andCDPK13genesaswell as truncated (VK) variants of the CDPK5, CDPK7, CDPK10, andCDPK13 geneswere prepared from the rice (cvNipponbare) cDNAbyPCRamplification with appropriate primers (Supplemental Table 3). Amino acidsubstitution of kinase-inactivated forms (K/M) of the CDPK5K/M (K121M)and CDPK13K/M (K117M) was introduced by PCR-based, site-directedmutagenesis. Then, the cDNA fragments were cloned into the pGreenbinary vector, and the cDNA fragment of the full-length RBOHH gene wascloned into the pGD binary vector as described by Kobayashi et al. (2007).Transformation of Agrobacterium (strain GV3101) and infiltration ofAgrobacterium intoN.benthamiana leavesweredoneasdescribedbyAsaiet al. (2008).

ROS production inN. benthamiana leaves wasmonitored by a luminol-based assay using L-012 (WakoPureChemical Industries) as described byKobayashi et al. (2007). Data were expressed as the average of the in-dicated number of replications. Two identical experiments were sub-sequently performed with comparable results.

Preparation of Protein Extracts and Immunoblotting

Microsomal proteins of N. benthamiana leaves were extracted as de-scribedbyKobayashi et al. (2007). Theproteinextractsweredenaturedandseparated by SDS-PAGE and then the gel was stained with CoomassieBrilliant Blue. For immunoblotting, FLAG-tagged and HA-tagged proteinswere detected by monoclonal anti-FLAG antibody (F3165; Sigma-Aldrich)and anti-HA antibody (clone HA-7; Sigma-Aldrich), respectively. Anti-mouse Ig antibody (GE Healthcare Life Sciences) and antigen complexwere detected using the ECL protein gel blot detection kit (GE HealthcareLife Sciences) and Light-Capture equipped with a CCD camera (ATTO) asdescribed by Kobayashi et al. (2007).

Generation and Analysis of Transgenic RBOHH-CRISPR/Cas9 Lines

To clarify the role of RBOHH in aerenchyma formation, RBOHH knockoutlines were generated using CRISPR/Cas9 (Feng et al., 2013; Shan et al.,2013; Mikami et al., 2015). The oligonucleotides for the RBOHH-CRISPR/Cas9 (RHC) vector construction (Supplemental Table 3) were annealedand cloned into the pZDgRNA_Cas9ver.2_HPT vector (Mikami et al.,2015) using the BbsI restriction site. Then the expression cassette onthe pZDgRNA_Cas9ver.2_HPT was replaced into the binary vector(pZH_gYSA_MMCas9) (Mikami et al., 2015) usingAscI andPacI restrictionsites. The guide RNA in the RHC vector was designed to induce a DNAdouble-strandbreakat10bpdownstreamof theATG initiationcodonof theRBOHH gene (Supplemental Figure 6A). The CRISPR/Cas9 vector withempty guide RNA was used as a vector control. The constructed vectorswere transformed into rice (cv Nipponbare) via Agrobacterium (strainEHA105), according to Toki et al. (2006).

Plants from each RHC line were grown under aerated conditions.Subsequently, three plants were transferred to aerated or stagnant con-ditions and grown for 48 h. To quantify aerenchyma formation, root crosssections of three root segments from each of three plants were preparedand photographed as described above. Subsequently, plants were grownfor another 5 d (a total of 1 week) and four root segments from two or threeplants were used to measure aerenchyma formation. For RT-qPCRanalysis and H2O2 quantification, segments of three roots from each ofthree plants were used for the experiments. Data were expressed as theaverage of the indicated number of replications.

786 The Plant Cell

Statistical Analysis

Statistical differences between means were calculated using two-samplet test. For multiple comparisons, data were analyzed by one-way ANOVAandpost hocTukey’s test usingSPSSStatisticsVersion19 (IBMSoftware).Results of statistical analyses are shown in Supplemental File 3.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdatabases under the accession numbers listed in Supplemental Table 4.

Supplemental Data

Supplemental Figure 1. Aerenchyma formation at 20 mm from thetips of rice roots.

Supplemental Figure 2. Aerenchyma formation and ethylene accu-mulation in rice roots.

Supplemental Figure 3. Phylogenetic analysis of RBOH proteins inplants.

Supplemental Figure 4. Effect of ruthenium red on aerenchymaformation in rice roots.

Supplemental Figure 5. Phylogenetic analysis of group I CDPKproteins in plants.

Supplemental Figure 6. Scheme of the CRISPR/Cas9-mediatedtarget mutagenesis of RBOHH, and genotyping of the transgenic lines.

Supplemental Figure 7. Aerenchyma formation in roots of RBOHH-targeted CRISPR/Cas9 transgenic lines.

Supplemental Figure 8. Aerenchyma formation in roots of RBOHH-targeted CRISPR/Cas9 transgenic lines under stagnant conditions for1 week.

Supplemental Figure 9. Effects of DPI and EGTA on RBOHHexpression in rice roots.

Supplemental Table 1. Number of seeds obtained from RBOHH-targeted CRISPR/Cas9 transgenic lines.

Supplemental Table 2. List of primers used for RT-qPCR analysis.

Supplemental Table 3. List of primers used for vector constructionand genotyping of transgenic plants.

Supplemental Table 4. List of sequence IDs in the GenBank/EMBLdatabase.

Supplemental File 1. Text file of the alignment used for the phyloge-netic analysis shown in Supplemental Figure 3.

Supplemental File 2. Text file of the alignment used for the phyloge-netic analysis shown in Supplemental Figure 5.

Supplemental File 3. Results of statistical analyses.

ACKNOWLEDGMENTS

WethankYoshiakiNagamura for providing informationongeneexpressionprofiles in the rice roots; Masaki Endo and Masafumi Mikami for theuniversal binary vector (CRISPR/Cas9); Kiyoaki Kato and Itsuro Takamurefor seeds of rice (cv Shiokari); Motoyuki Ashikari for the protocol of theethylene collection apparatus; Phil Mullineaux and Roger Hellens forpGreen vector; Andrew O. Jackson for pGD binary vector; Rohm andHaas Japan and AgroFresh for 1-MCP; and the Leaf Tobacco ResearchCenter, Japan, for N. benthamiana seeds. We also thank Timothy

D.Colmer,OlePedersen, andAl ImranMalik for critical suggestions, aswellas Katsuhiro Shiono, Hirokazu Takahashi, Kazuyuki Kuchitsu, TakamitsuKurusu, Kenji Hashimoto, Atsushi Oyanagi, Kentaro Kawaguchi, FumitakaAbe, YoshiroMano,Mitsuhiro Obara, Tomomi Abiko, Shunsaku Nishiuchi,and Kohtaro Watanabe for stimulating discussion. This work was partlysupported by a grant from the Bio-oriented Technology Research Ad-vancement Institution (Promotion of Basic Research Activities for Innova-tive Biosciences) to M.N. H.Y. was supported by a Grant-in-Aid forScientific Research on Innovative Areas “Oxygen Biology: a new criterionfor integrated understanding of life” (15H01398) from MEXT of Japan andScientificResearch (26292023) from the JapanSociety of thePromotion ofScience. T.Y. was supported by a postdoctoral fellowship from the JapanSociety for the Promotion of Science.

AUTHOR CONTRIBUTIONS

T.Y. and M.N. designed the experiments and wrote the article. M.N.supervised the experiments. M.Y. performed ROS detection experimentsusingN.benthamiana leaves under supervision ofH.Y. H.M. performed theethylene measurements. T.Y. performed the rest of the experiments withthe contribution of A.F. N.K.N., N.T., and H.Y. provided advice on theresearch plans.

Received December 30, 2016; revisedMarch 6, 2017; acceptedMarch 24,2017; published March 28, 2017.

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790 The Plant Cell

DOI 10.1105/tpc.16.00976; originally published online March 28, 2017; 2017;29;775-790Plant Cell

Tsutsumi, Hirofumi Yoshioka and Mikio NakazonoTakaki Yamauchi, Miki Yoshioka, Aya Fukazawa, Hitoshi Mori, Naoko K. Nishizawa, Nobuhiro

under Oxygen-Deficient ConditionsAn NADPH Oxidase RBOH Functions in Rice Roots during Lysigenous Aerenchyma Formation

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