Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting...

6
Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress Daisuke Todaka a,b , Kazuo Nakashima a , Kyonoshin Maruyama a , Satoshi Kidokoro b , Yuriko Osakabe b , Yusuke Ito a , Satoko Matsukura a,b , Yasunari Fujita a , Kyouko Yoshiwara a , Masaru Ohme-Takagi c , Mikiko Kojima d , Hitoshi Sakakibara d , Kazuo Shinozaki e , and Kazuko Yamaguchi-Shinozaki a,b,1 a Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan; b Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; c Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan; and d Plant Productivity Systems Research Group and e Gene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan Edited* by Steve A. Kay, University of California at San Diego, La Jolla, CA, and approved August 16, 2012 (received for review May 1, 2012) The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-inter- acting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was ob- served in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis re- vealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data sug- gest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adap- tation in rice under drought conditions. abiotic stress | plant height regulation D rought is the biggest constraint of growth and development in plants, often resulting in reduced crop yields (1, 2). To produce crops with high yields even under drought conditions, it is essential to unravel the molecular mechanisms of growth re- duction in response to drought stress. Growth reduction under stress conditions has been considered a secondary effect of re- duced photosynthetic activity and stomatal closure (3). However, it is now accepted that plants actively reduce their growth to save photosynthetic resources and decrease transpiration area as a stress adaptation response (4). Under stress conditions, plants induce expression of a wide variety of genes. Among these genes, key factors that function in stress responses have been identied (58). These factors include: DRE binding 1 (DREB1)/C-repeat binding factor-type transcrip- tion factors that are involved in response to low-temperature stress; DREB2-type transcription factors that play important roles in drought and high-temperature stress-responsive gene expres- sion; ABA responsive element binding /ABA binding factor-type transcription factors that modulate the ABA-dependent transcrip- tional network; and NAC-type transcription factors that are in- volved in response to drought and high-salinity stresses. These factors are known to function as transcriptional activators to en- hance abiotic stress tolerance. Constitutive expression of these transcription factor genes in transgenic Arabidopsis or rice confers tolerance to abiotic stresses (911). Moreover, overexpression of these genes causes growth reduction phenotypes in transgenic plants, but the molecular mechanisms of these phenotypes remain unclear. Knowledge of physiological and molecular mechanisms for growth reduction in response to abiotic stresses is still fragmented. Drought decreases leaf area by reducing both cell number and size (3, 4, 12). This reduced cell number is attributed to the inhibition of the G1-to-S transition, mediated by a specic cyclin-dependent kinase (4, 13). Cell size modulation under stress conditions is ac- companied by a change in cell wall rheology via cell wall-related genes, including expansins (3, 14, 15). Key factors that function in the transcriptional network of growth reduction in response to abiotic stresses are still unclear. To dissect transcriptional networks in abiotic stress responses, it is important to study transcription factors that are differentially ex- pressed between stressed and nonstressed plants. Although most transcription factors that function in stress tolerance are up-regu- lated by abiotic stresses, we detected candidate transcription factors down-regulated by these stresses. Here, we report a gene for a phy- tochrome (phy)-interacting basic helix-loop-helix transcription factor (PIF)-like protein down-regulated under drought stress conditions. One of the PIF family transcription factors was initially isolated as a protein that interacted with phytochrome through a yeast two-hy- brid screen (16, 17). PIFs have been shown to be components of various developmental responses, including seed germination, seedling growth, and cell fate (18). We propose that the rice PIF-like protein can act as an internodal growth regulator and plays an im- portant role in a drought-associated growth-restriction mechanism that functions as a morphological adaptation to drought stress. Results Expression Patterns of OsPIL1. A rice PIF-like gene (OsPIL1/ OsPIL13, LOC_Os03g56950) was identied by microarray anal- yses (19) as one of the stress-responsive transcription factor genes that were down-regulated by drought stress. First, we conrmed the stress-responsive expression pattern of the OsPIL1 gene. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with 12-h light/12-h dark cycles oscillated in a circadian manner, with peaks in the middle of the light period (Fig. 1A). Author contributions: D.T., K.N., Y.F., K.S., and K.Y.-S. designed research; D.T., K.M., S.K., Y.O., Y.I., S.M., K.Y., and M.K. performed research; M.O.-T. contributed new reagents/ analytic tools; D.T., K.M., and H.S. analyzed data; and D.T. and K.Y.-S. wrote the paper. The authors declare no conict of interest. *This Direct Submission article had a prearranged editor. Data deposition: The microarray data have been deposited in the European Bioinfor- matics Institute ArrayExpress database, www.ebi.ac.uk/arrayexpress (accession no. E-MEXP-3605). 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1207324109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1207324109 PNAS | September 25, 2012 | vol. 109 | no. 39 | 1594715952 PLANT BIOLOGY Downloaded by guest on November 18, 2020

Transcript of Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting...

Page 1: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

Rice phytochrome-interacting factor-like protein OsPIL1functions as a key regulator of internode elongationand induces a morphological response to drought stressDaisuke Todakaa,b, Kazuo Nakashimaa, Kyonoshin Maruyamaa, Satoshi Kidokorob, Yuriko Osakabeb, Yusuke Itoa,Satoko Matsukuraa,b, Yasunari Fujitaa, Kyouko Yoshiwaraa, Masaru Ohme-Takagic, Mikiko Kojimad,Hitoshi Sakakibarad, Kazuo Shinozakie, and Kazuko Yamaguchi-Shinozakia,b,1

aBiological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan; bLaboratoryof Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; cGene Function ResearchCenter, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8562, Japan; and dPlant Productivity Systems Research Groupand eGene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan

Edited* by Steve A. Kay, University of California at San Diego, La Jolla, CA, and approved August 16, 2012 (received for review May 1, 2012)

Themechanisms for plant growth restriction during stress conditionsremains unclear. Here, we demonstrate that a phytochrome-inter-acting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator ofreduced internode elongation in rice under drought conditions. Thelevel of OsPIL1 mRNA in rice seedlings grown under nonstressedconditions with light/dark cycles oscillated in a circadian mannerwith peaks in the middle of the light period. Under drought stressconditions, OsPIL1 expression was inhibited during the light period.We found that OsPIL1was highly expressed in the node portions ofthe stem using promoter-glucuronidase analysis. Overexpression ofOsPIL1 in transgenic rice plants promoted internode elongation. Incontrast, transgenic rice plants with a chimeric repressor resulted inshort internode sections. Alteration of internode cell size was ob-served in OsPIL1 transgenic plants, indicating that differences in cellsize cause the change in internode length. Oligoarray analysis re-vealed OsPIL1 downstream genes, which were enriched for cellwall-related genes responsible for cell elongation. These data sug-gest thatOsPIL1 functions as a key regulatory factor of reduced plantheight via cell wall-related genes in response to drought stress. Thisregulatory systemmay be important for morphological stress adap-tation in rice under drought conditions.

abiotic stress | plant height regulation

Drought is the biggest constraint of growth and developmentin plants, often resulting in reduced crop yields (1, 2). To

produce crops with high yields even under drought conditions, itis essential to unravel the molecular mechanisms of growth re-duction in response to drought stress. Growth reduction understress conditions has been considered a secondary effect of re-duced photosynthetic activity and stomatal closure (3). However,it is now accepted that plants actively reduce their growth to savephotosynthetic resources and decrease transpiration area as astress adaptation response (4).Under stress conditions, plants induce expression of a wide

variety of genes. Among these genes, key factors that function instress responses have been identified (5–8). These factors include:DRE binding 1 (DREB1)/C-repeat binding factor-type transcrip-tion factors that are involved in response to low-temperature stress;DREB2-type transcription factors that play important roles indrought and high-temperature stress-responsive gene expres-sion; ABA responsive element binding /ABA binding factor-typetranscription factors that modulate the ABA-dependent transcrip-tional network; and NAC-type transcription factors that are in-volved in response to drought and high-salinity stresses. Thesefactors are known to function as transcriptional activators to en-hance abiotic stress tolerance. Constitutive expression of thesetranscription factor genes in transgenicArabidopsis or rice conferstolerance to abiotic stresses (9–11). Moreover, overexpression of

these genes causes growth reduction phenotypes in transgenic plants,but the molecular mechanisms of these phenotypes remain unclear.Knowledge of physiological and molecular mechanisms for

growth reduction in response to abiotic stresses is still fragmented.Drought decreases leaf area by reducing both cell number and size(3, 4, 12). This reduced cell number is attributed to the inhibitionof the G1-to-S transition, mediated by a specific cyclin-dependentkinase (4, 13). Cell size modulation under stress conditions is ac-companied by a change in cell wall rheology via cell wall-relatedgenes, including expansins (3, 14, 15). Key factors that function inthe transcriptional network of growth reduction in response toabiotic stresses are still unclear.To dissect transcriptional networks in abiotic stress responses, it is

important to study transcription factors that are differentially ex-pressed between stressed and nonstressed plants. Although mosttranscription factors that function in stress tolerance are up-regu-lated by abiotic stresses, we detected candidate transcription factorsdown-regulated by these stresses. Here, we report a gene for a phy-tochrome (phy)-interacting basic helix-loop-helix transcription factor(PIF)-like protein down-regulated under drought stress conditions.One of the PIF family transcription factors was initially isolated asa protein that interacted with phytochrome through a yeast two-hy-brid screen (16, 17). PIFs have been shown to be components ofvarious developmental responses, including seed germination,seedling growth, and cell fate (18).We propose that the rice PIF-likeprotein can act as an internodal growth regulator and plays an im-portant role in a drought-associated growth-restriction mechanismthat functions as a morphological adaptation to drought stress.

ResultsExpression Patterns of OsPIL1. A rice PIF-like gene (OsPIL1/OsPIL13, LOC_Os03g56950) was identified by microarray anal-yses (19) as one of the stress-responsive transcription factor genesthat were down-regulated by drought stress. First, we confirmedthe stress-responsive expression pattern of the OsPIL1 gene. Thelevel of OsPIL1 mRNA in rice seedlings grown under nonstressedconditions with 12-h light/12-h dark cycles oscillated in a circadianmanner, with peaks in the middle of the light period (Fig. 1A).

Author contributions: D.T., K.N., Y.F., K.S., and K.Y.-S. designed research; D.T., K.M., S.K.,Y.O., Y.I., S.M., K.Y., and M.K. performed research; M.O.-T. contributed new reagents/analytic tools; D.T., K.M., and H.S. analyzed data; and D.T. and K.Y.-S. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The microarray data have been deposited in the European Bioinfor-matics Institute ArrayExpress database, www.ebi.ac.uk/arrayexpress (accession no.E-MEXP-3605).1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207324109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207324109 PNAS | September 25, 2012 | vol. 109 | no. 39 | 15947–15952

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0

Page 2: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

Intriguingly, when drought stress started in the middle of the darkperiod, OsPIL1 expression was not elevated during the light pe-riod. When drought stress started early in the light period,OsPIL1expression was drastically decreased to a level similar to thatobserved in the dark period. Cold stress also inhibited expressionduring the light period (Fig. 1B). High-salinity (NaCl), high-temperature, ABA and gibberellic acid (GA) did not obviouslychange the OsPIL1 expression pattern. When the rice seedlingswere treated with ethephon, OsPIL1 expression reached a peakearlier than in the control plants.

Histochemical Analysis and Subcellular Localization of OsPIL1. Pro-moter-glucuronidase (GUS) analyses were performed to revealhistological localization of OsPIL1 expression. GUS signals inseedlings were detected in the leaves and the basal part of shoots(Fig. 2A). At the early heading stage, GUS signals were observedin the nodal regions, especially in the nodal septum (Fig. 2 B andC). To confirm these GUS signals at the heading stage, themRNA levels of OsPIL1 were investigated using quantitativeRT-PCR. In agreement with the promoter-GUS analyses, muchhigher OsPIL1 mRNA levels were detected in the node regioncompared with the internode region (Fig. 2D).To examine the subcellular localization of OsPIL1 protein, we

performed a transient expression assay using rice protoplast cellsprepared from shoots. Clear GFP-tagged OsPIL1 protein signalswere detected in the nucleus (Fig. S1A), suggesting that the OsPIL1protein was localized to the nucleus. We also examined transgenicrice seedlings expressing GFP-tagged OsPIL1. GFP signals weredetected in the nucleus of cells in the basal part of shoots (Fig. S1C).

Morphological Phenotypes of Transgenic Plants Overexpressing OsPIL1or Expressing an OsPIL1 Chimera Repressor. To further elucidate thefunction of the OsPIL1 protein, we generated transgenic rice

plants overexpressing OsPIL1 (OsPIL1-OXs) or expressingOsPIL1 fused to a repression domain (RD) that consists of only12 amino acids to convert a transcriptional activator into a tran-scriptional repressor and to overcome potential functional re-dundancy conferred by homologs (OsPIL1-RDs) (20).We obtainedseven independent OsPIL1-OX candidates and six independentOsPIL1-RD candidates, and selected transformants with hightransgene expression levels for further analyses (OsPIL1-OX#2,OsPIL1-OX#17, OsPIL1-RD#3, and OsPIL1-RD#5) (Fig. S2A).Although we also tried to generate RNAi transgenic rice, we couldnot obtain transformants in which the expression level of OsPIL1was reduced. OsPIL1-OX and OsPIL1-RD rice seedlings showeda slight increase and decrease in plant height, respectively (Fig. S2B and C). At 30 d after imbibition, OsPIL1-OX and OsPIL1-RDrice plants showed a significant increase and decrease, respectively,in stem length between the shoot base and the uppermost laminajoint (Fig. S2 D and E). At the adult stage, OsPIL1-OXs displayedenhanced stem elongation, leading to a strikingly tall plant (Fig. 3Aand C and Fig. S2I). This result was mainly because of increasedelongation in each internode (Fig. S2J). In contrast, adult OsPIL1-RD plants were shorter than vector control plants. The sheathlengths of flag leaves were not different between vector controlplants and OsPIL1-OXs orOsPIL1-RDs (Fig. S2G). Consequently,in OsPIL1-OXs the sheath length was shorter than the internodelength (Fig. S2 F,H, and K). In OsPIL1-RDs the sheath length wasgreater than the internode length, resulting in imperfect emergenceof panicles (Fig. S2 F, H, and K).To determine whether the abnormal internode elongation was

caused by alterations in cell number or cell size, longitudinalsections of internodes were examined. The first internode cells ofOsPIL1-OXs were larger than those of nontransgenic controlplants (Fig. 3 B and D). Smaller internode cells were found inOsPIL1-RDs. These results suggest that the differences in in-ternode lengths between control plants and OsPIL1-OXs orOsPIL1-RDs primarily resulted from alterations in cell size. Thevertical seed lengths were longer in OsPIL1-OXs and slightlyshorter in OsPIL1-RDs compared with those of nontransgenic

Fig. 1. Expression analysis of OsPIL1 by quantitative RT-PCR. Bars indicatethe SD of three to four replicates. Arrows indicate the start point of thestress treatment. (A) Expression patterns of OsPIL1 under nonstress (control)or drought stress (drought) conditions. (B) Expression patterns of OsPIL1when rice seedlings were kept at 4 °C (cold) or 37 °C (hot), or the roots wereimmersed in 250 mM NaCl, 25 μM ABA, 25 μM GA, or 100 μM ethephon.

Fig. 2. Promoter-GUS staining analysis of OsPIL1. Similar expression pat-terns were observed in multiple independent transgenic lines. (A) Expressionof the GUS gene in 2-wk-old seedlings. (Upper) The leaf region. (Lower) Alongitudinal section at the basal part of the seedling. (Scale bar, 2 mm.) (B)Expression of GUS in the node and internode portions of the stem atheading stage. Photos show longitudinal sections. (Scale bars, 20 mm.) (C)Expression of GUS in the node portion of the stem at heading stage. (Scalebars, 5 mm.) ls, leaf sheath; lsp, leaf-sheath pulvinus; mc, medullary cavity;ns, nodal septum. (D) Quantitative RT-PCR analysis of OsPIL1 expression inpanicles, first internodes, first nodes, and second internodes at headingstage. Bars indicate the SD of three to four replicates.

15948 | www.pnas.org/cgi/doi/10.1073/pnas.1207324109 Todaka et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0

Page 3: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

control plants (Fig. S2 L and M). The yield was not significantlydifferent between the transgenic plants and the vector controls(Fig. S2N).Transgenic Arabidopsis plants overexpressing OsPIL1 (35S:

OsPIL1) showed longer hypocotyls than vector control plants (Fig.S2 O and P). In contrast, transgenic Arabidopsis plants expressingOsPIL1 fused to the RD (35S:OsPIL1:RD) had short hypocotyls.At the adult stage, the 35S:OsPIL1 and 35S:OsPIL1:RD plantsshowed no increase or decrease in plant height (Fig. S2Q).

Downstream Genes of OsPIL1. To identify downstream genes reg-ulated by the OsPIL1 transcription factor, we performed amicroarray analysis using the OsPIL1 transgenic rice plants.Because OsPIL1-OXs showed increased internode elongation(Fig. 3) and the OsPIL1 gene was highly expressed in the firstnode portion (Fig. 2), only the first node portions of OsPIL1-OXs and vector control plants were used for the microarrayanalysis. After RNA extraction from the first node portion, large-scale expression profiles were compared between OsPIL1-OXsand vector control plants. The transcriptome analysis identified1,396 genes up-regulated [formal concept analysis (FCA) > 2.0]and 1,358 genes down-regulated (FCA < 2.0) in the first nodeportion of OsPIL1-OXs (Dataset S1). Expression of more thanhalf of the up-regulated genes was decreased under droughtstress (790 of 1,396 genes), and expression of large numbers ofthe down-regulated genes was increased by drought stress (480 of1,358 genes) in our microarray data (Fig. 4A). A gene-expressionsearch engine, GENEVESTIGATOR, also confirmed the de-creased expression of the OsPIL1-OX up-regulated genes underdrought conditions (Fig. 4B).To obtain an ontological profile of the up- or down-regulated

genes in OsPIL1-OXs, ontological terms were assigned (Fig. S3A),and the enrichment significance was analyzed by AgriGO. SixteenGene Ontology (GO) terms were significantly enriched in theup-regulated gene set (Table S1). Of the enriched GO terms, wenoted ones related to cell wall development because activatedcell elongation was observed in OsPIL1-OXs (Fig. 3 B and D).Four GO terms were related to cell wall development: cell wallorganization or biogenesis (GO:0071554), cell wall organiza-tion (GO:0071555), cellulose metabolic process (GO:0030243),and cellulose biosynthetic process (GO:0030244). These GOsincluded 39 genes, such as expansins and cellulose synthases (TableS2). Most of these genes were down-regulated under droughtconditions (Table S2). Furthermore, we investigated gene-ex-pression levels in nonstressed and drought-stressed node por-tions. In drought-stressed node portions, the expression levelsof Expansin S1 (Os03g0336400), Expansin S2 (Os04g0228400),OsEXPA2 (Os01g0823100), OsEXPA4 (Os05g0477600), and Ex-tensin protein-like (Os03g0637600), as well as OsPIL1, werelower than in the nonstressed node portions (Fig. 4C). Higherexpression levels of OsNAC6 (21) and OsDREB2B2 (22) indrought-stressed node portions compared with nonstressed onessupported the effectiveness of this stress manipulation. Thesedata suggest that the cell wall-related genes identified here areideal candidates for OsPIL1 downstream genes.We searched for cis-element candidates in the upstream

regions of the OsPIL1 downstream gene ORFs using RiCES.CACGTG (G-box) sequences, which have been shown to berecognition sites for Arabidopsis PIF proteins (23), were found inthe upstream regions of OsPIL1 downstream genes (Table S3).

Fig. 3. Phenotypes of OsPIL1 transgenic rice plants. (A) Morphology ofmature vector control plants (Con), transgenic rice plants overexpressingOsPIL1 (OX), and plants expressing an OsPIL1 chimeric repressor that con-tains an additional repression domain (RD). Some leaves and leaf sheathswere removed to check the position of each node. PN, panicle node; 1N, firstnode; 2N, second node; 3N, third node. (Scale bars, 30 cm.) (B) Longitudinalsections at the first internodes in mature Con, OX, and RD plants. The leftside of each photo indicates silicified epidermis (se) and sclerenchymatous

fiber tissue (sft), and the right edge shows a central lacuna (cl). (Scale bars,0.1 μm.) (C) Heights of mature Con, OX, and RD plants. **P < 0.001 com-pared with the control value in Scheffé’s test. Bars indicate the SD of 7–17replicates. (D) Vertical lengths of parenchyma cells in Con, OX, and RDplants. **P < 0.001 compared with the control value in Scheffé’s test. Barsindicate the SD of 30 replicates.

Todaka et al. PNAS | September 25, 2012 | vol. 109 | no. 39 | 15949

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0

Page 4: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

Other sequences were also detected, including CAAT[GC]ATTG and CCA[ACTG]TG, which are binding sites for HD-ZIP and LEAFY, suggesting that these transcription factorsmight be also involved in the OsPIL1 downstream cascade.

Transactivation Activity of OsPIL1. To verify whether OsPIL1 func-tions as a transcriptional regulator, we investigated the transacti-vation activity of OsPIL1 using the transient assay system (22, 24).Protoplasts were cotransfected with GUS reporter constructscontaining the promoter region from OsEXPA4 (Os05g0477600),which was identified above as one of the OsPIL1 downstreamgenes, and effector plasmids containing OsPIL1 cDNA fused to theubiquitin promoter (Fig. 4D). In the presence of the effector, the−1 kb OsEXPA4 promoter (Long Exp) increased GUS activity(Fig. 4E). This promoter region contained a copy of the G-boxelement. A short OsEXPA4 promoter (Short Exp) containing theG-box element similarly increased GUS activity. A promoterlacking the G-box element (Del G Exp) showed decreased GUSactivity compared with Long Exp or Short Exp. When a constructcontaining additional copies of the G-box element (G3 Exp) wasused, GUS activity was higher than in Long Exp or Short Exp.Furthermore, we analyzed the promoter of another gene, 1-amino-cyclopropane-1-carboxylate (1-ACC) oxidase (Os09g0451400),because we predicted that it was one of the downstream candidategenes of OsPIL1. This gene was up-regulated in the OsPIL1-OXs(FCA = 3.5) (Dataset S1) and down-regulated in the drought-stressed node (Fig. 4C). We obtained similar data to the OsEXPA4promoter using the GUS gene fused to the −1 kb 1-ACC oxidasepromoter (Fig. S3C). These results suggested that OsPIL1 couldactivate expression of the OsEXPA4 and 1-ACC oxidase genes viathe G-box element. To check whether the OsPIL1-GFP proteinwas functional, we performed a transactivation assay using the G3Exp promoter-GUS construct. The reporter GUS activity wasenhanced by the addition of OsPIL1-GFP proteins, suggestingthat the fusion protein was functional (Fig. S3E).We also examined the effect of OsPhyB on the transcriptional

activity of OsPIL1 using the transient reporter assay. Addition ofOsPhyB did not change the GUS activity under either dark orlight conditions, whereas PhyB decreased the transcriptionalactivity of PIF4 (Fig. S3F). This finding suggested that OsPhyBdoes not affect the transcriptional activity of OsPIL1. This hy-pothesis was supported by the observation that OsPIL1 did notinteract with OsPhyB in the yeast two-hybrid assay (Fig. S3G) orthe bimolecular fluorescence complementation assay (Fig. S3H),and by the fact that one of the four residues important for PhyBbinding in Arabidopsis PIFs (25) was not conserved in OsPIL1(Fig. S3I).

Hormone Content.Growth modulation by PIF4 was reported to bemediated by indole-3-acetic acid (26) or GA (27) signaling. Tocheck the involvement of plant hormones in OsPIL1-regulatedstem elongation, we measured hormone content in the OsPIL1transgenic rice plants (Table S4). We used growing stem regionsfrom the shoot base to the uppermost lamina joint at 30 d afterimbibition as materials (Fig. S2 D and E). The levels of GA1,GA19, GA20, GA44, and GA53 were slightly decreased inOsPIL1-OXs but not significantly changed in OsPIL1-RDs. Thelevels of other hormones were not significantly different betweenthe OsPIL1 transgenic rice plants, suggesting that the hormonesmeasured here were not involved in the regulation of stemelongation by OsPIL1. Thus, OsPIL1 internode elongation maybe modulated by a different pathway from Arabidopsis PIF4.

DiscussionThe molecular mechanisms for the regulation of plant growthand development during stress conditions remains unclear. Ourdata provide evidence that OsPIL1 functions as a key regulatorof reduced plant height in rice during stress conditions. Several

Fig. 4. Downstream genes of OsPIL1. (A) Expression profiles of the OsPIL1downstream genes by GENEVESTIGATOR (https://www.genevestigator.com/gv/user/gvLogin.jsp). The responses of the up-regulated (FCA > 2.0) genes inthe node portion of OsPIL1 OX plants to external stimuli or perturbationswere analyzed. (B) Expression changes under drought conditions of up-(FCA > 2.0) or down- (FCA < 2.0) regulated genes in the node portion ofOsPIL1 OX plants, from our earlier microarray data (19). (C) Expression levelsof OsPIL1, the representative OsPIL1 downstream genes (Expansin S1, Expan-sin S2, OsEXPA2, OsEXPA4, Extensin, and 1-ACC oxidase) and drought-in-ducible marker genes (OsNAC6 and OsDREB2B2) in the node portions ofwild-type rice plants at heading stage under nonstressed (Con) or drought-stressed (DS) conditions by quantitative RT-PCR. Wild-type rice plants atheading stage were subjected to drought stress for 40 h after uprooting.Bars indicate the SD of three to five replicates. (D) Design of constructs fortransient expression assay. (E) Transient expression assay using rice pro-toplast cells. Intrinsic long promoters (−1,000 to −1 bp, designated LongExp), intrinsic short promoters (G-box location to −1 bp, designated ShortExp), intrinsic G-box-deleted promoters (fragments without a G-box, desig-nated Del G Exp), and artificial promoters that include three G-box elements(designated G3 Exp) were used. Constructs containing the LUC gene drivenby the ubiquitin promoter were used to normalize transfection efficiency.Bars indicate the SD of five to seven replicates.

15950 | www.pnas.org/cgi/doi/10.1073/pnas.1207324109 Todaka et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0

Page 5: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

results support this conclusion: (i) OsPIL1-OXs showed acti-vated internode elongation via increased cell size, whereasOsPIL1-RDs had short internode sections resulting from de-creased cell size (Fig. 3); (ii) OsPIL1 downstream genes includeda number of the cell wall-related genes (Table S2), which havebeen reported to be involved in plant growth regulation by cellelongation (28, 29); and (iii) light-dependent expression ofOsPIL1 was clearly inhibited during stress conditions (Fig. 1).The activated internode elongation observed in OsPIL1-OXs

is the most intriguing phenotype (Fig. 3 A and C). Internodeelongation is considered to be caused by two kinds of regulatorymechanisms (15). One mechanism is an enhanced cell-divisionrate in the meristematic region, initiated by GA-induced ex-pression of cyclin genes and a p34cdc2-like histone H1 kinase(15, 30). The other mechanism is cell elongation achieved byregulation of microtubule orientation, creep of cell-wall poly-mers, and biosynthesis, transport, and incorporation of new cell-wall components (15, 31). We concluded that cell elongationcaused the activated internode elongation in OsPIL1-OXs, be-cause of alterations in internode cell size (Fig. 3 B and D). Thisconclusion was further supported by the finding that the set ofup-regulated genes in OsPIL1-OXs included a number of genesassociated with cell wall biosynthesis and development (TableS2). Expansins have been shown to regulate cell elongation bycell wall expansion, which is caused by acid-induced cell wallrelaxation (15, 32–34). α-Expansin OsEXPA4 overexpressors showan elongated shoot, coleoptile, and mesocotyl, resulting fromchanges in cell size (29). A network of extensins is thought to beinvolved in the control of cell elongation and cell-wall architec-ture (35, 36). Cellulose synthase is also involved in cell wall for-mation and plant growth. A rice cellulose synthase mutant showedsignificant reduction in plant growth (37). These factors reinforcethe possibility that the cell wall-related genes identified here asOsPIL1 downstream genes caused the abnormal internode elon-gation in OsPIL1 transgenic rice plants. Most of the up-regulatedgenes in OsPIL1-OXs were significantly expressed in aerobic andanaerobic seed germination (Fig. 4B), which suggests that theyare involved in germination processes. Germination processescause cell proliferation and enlargement, and are adversely af-fected by drought conditions.The set of down-regulated genes in OsPIL1-OXs included

many drought-inducible genes in rice plants (Fig. 4A). Wecompared these down-regulated genes with genes downstream ofthe transcription factors OsDREB1A, OsNAC6, and OsbZIP23,which have been reported to positively regulate expression ofstress-responsive genes in rice plants. The number of genesdownstream of OsDREB1A, OsNAC6, and OsbZIP23 was 81,158, and 743, respectively (38–40). Among these genes, only 13,37, and 61 genes overlapped with the down-regulated genes inOsPIL1-OXs, respectively. These results suggest that the path-ways regulated by OsDREB1A, OsNAC6 and OsbZIP23 act inparallel to the pathway regulated by OsPIL1.The OsPIL1 mRNA level in rice seedlings grown under non-

stressed conditions with light/dark cycles oscillated in a circadianmanner with peaks in the middle of the light period (Fig. 1). Thisfinding is similar to Arabidopsis PIF4 and PIF5 expression patterns(41). Phylogenetically, Arabidopsis PIF4 and PIF5 are the closestorthologs of OsPIL1 (38). Transgenic Arabidopsis plants over-expressing PIF4 or PIF5 showed increased hypocotyl elongation(42, 43). We observed a similar morphological phenotype intransgenicArabidopsis seedlings overexpressingOsPIL1 (Fig. S2Oand P). These results imply that rice OsPIL1 may have a similarrole to Arabidopsis PIF4 and PIF5 in plant developmental pro-cesses. PIF4 was shown to control photoperiodic growth (41, 44),stomatal development under high light intensity (45), and thermalacceleration of flowering (46) and petiole elongation (47). Thesestudies suggest that PIF4 functions as a node that connects de-velopment with external stimuli such as light and temperature.

Arabidopsis PIF4 and PIF5 have also been shown to functionin shade avoidance (43). These transcription factors accumulatein the dark and act as constitutive repressors of photomorpho-genesis in seedlings. In response to red light these proteins areselectively degraded, which requires their interaction with light-activated PhyB. The proteins are stable in the shade and re-quired for shade avoidance that promotes shade-induced accel-eration of hypocotyl elongation (44). In contrast to ArabidopsisPIFs, OsPIL1 was shown not to interact with OsPhyB (Fig. S3),suggesting that OsPIL1 is more stable and active upon exposureto light than Arabidopsis PIFs and may affect plant growth morestrongly. However, as the OsPIL1 mRNA level was decreased bydrought stress, internode elongation does not occur under thestressed conditions in rice. This strategy gives plants an advan-tage in that chances of shading by neighbor plants are signifi-cantly increased. Shading provides plants with lower temperatureconditions, reducing transpiration rate and increasing surviv-ability when plants are rewatered. A similar strategy has beenreported in the response to UV-B exposure. UV-B decreasesstem elongation, increasing opportunities to use shade providedby neighbor plants (48, 49). Because the exogenousOsPIL1 expres-sion in OsPIL1-OXs is expected to be constitutive even underabiotic stress conditions, morphological studies of OsPIL1-OXsunder the stress conditions may provide a better understandingof the physiological function of OsPIL1 in rice.In conclusion, our data provide a model for OsPIL1 regulation

of plant height via cell wall-related genes under drought stressconditions (Fig. S4). Under nonstress conditions, light exposureincreases the expression of OsPIL1. This expression leads toincreased expression of cell wall-related genes, and consequentlycell elongation, resulting in internode elongation. Conversely,drought stress decreases the expression of OsPIL1 under lightillumination, leading to decreased expression of cell wall-relatedgenes. This process inhibits cell elongation, resulting in dwarfheight. Down-regulation of OsPIL1 expression may function asa reliable morphological adaptation system for reducing plantheight under drought stress conditions.

Materials and MethodsPlant Materials and Stress Treatments. Rice (Oryza sativa cv. Nipponbare)plants were grown as described previously (21, 39). Drought, high-salinityand low-temperature stress experiments were conducted as described in ref.21. A high-temperature stress experiment was performed as described in ref.22). Ethephon was used as an alternative to ethylene.

Generation of Transgenic Plants. To develop OsPIL1-OXs, we inserted theOsPIL1 cDNA ORF into a plant expression vector (50) containing a ubiquitinpromoter region (51). This construct was introduced into wild-type rice cv.Nipponbare by Agrobacterium-mediated transformation (52). OsPIL1-RDswere generated using the CRES-T system (20, 53). To develop transgenic riceplants expressing a GUS gene driven by the OsPIL1 promoter, we insertedthe OsPIL1 promoter region into a GUS expression vector and introduced theconstruct into rice plants (21)

Transient Expression in Rice Mesophyll Protoplasts. Protoplasts from riceshoots were prepared as described in ref. 54. PEG-calcium transfection wasperformed for transient expression assay (54). After incubation, reporteractivity was measured as described previously (22, 24).

Microarray Analysis. Total RNA for microarray analysis was isolated from nodeportions of OsPIL1-overexpressing and vector control plants at the headingstage. Themicroarray analysiswas performed as described in ref. 19.Microarraydata are available at the European Bioinformatics Institute (http://www.ebi.ac.uk/microarray-as/ae), with the accession number E-MEXP-3605.

Quantitative RT-PCR Analysis. Total RNA was prepared using RNAiso reagent(Takara Bio). PCR product levels were normalized by the expression value of18S rRNA as an internal standard. The OsPIL1 primers were 5′-GCAAACAG-TGCCACCACAGG-3′ (1,092–1,111 nt from ATG) and 5′-CTAAATTCCATCAG-

Todaka et al. PNAS | September 25, 2012 | vol. 109 | no. 39 | 15951

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0

Page 6: Rice phytochrome-interacting factor-like protein OsPIL1 ... · Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces

AGGTTGG-3′ (1,213–1,233 nt from ATG). The 18S rRNA primers were 5′-AT-GGTGGTGACGGGTGAC-3′ and 5′-CAGACACTAAAGCGCCCGGTA-3′.

Measurement of Plant Hormone Levels. The plant hormone levels in 100 mg oftissue from each plant was quantified as described in ref. 55 using a liquidchromatography-MS system (UPLC/Quattro Premier XE; Waters) with an ODScolumn (AQUITY-UPLC BEH-C18, 1.7 μm, 2.1 × 50 mm). Three biologicalreplicates were used in each experiment.

ACKNOWLEDGMENTS. We thank E. Ohgawara, K. Murai, K. Amano, andE. Kishi for their excellent technical support, and M. Toyoshima for skillfuleditorial assistance. This work was supported by grants from the Ministry ofAgriculture, Forestry and Fisheries of Japan (in part by Genomics forAgricultural Innovation, Development of Abiotic Stress Tolerant Crops byDREB Genes) and the Programme for Promotion of Basic and AppliedResearches for Innovations in Bio-oriented Industry of Japan. The hor-mone analysis reported here was supported by Japan Advanced PlantScience Network.

1. Boyer JS (1982) Plant productivity and environment. Science 218:443–448.2. Boyer JS, Westgate ME (2004) Grain yields with limited water. J Exp Bot 55:2385–2394.3. Skirycz A, Inzé D (2010) More from less: Plant growth under limited water. Curr Opin

Biotechnol 21:197–203.4. Skirycz A, et al. (2010) Developmental stage specificity and the role of mitochondrial

metabolism in the response of Arabidopsis leaves to prolonged mild osmotic stress.Plant Physiol 152:226–244.

5. Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks incellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol57:781–803.

6. Cominelli E, Tonelli C (2010) Transgenic crops coping with water scarcity. New Bio-technol 27:473–477.

7. Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory net-works in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149:88–95.

8. Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58.

9. Sakuma Y, et al. (2006) Functional analysis of an Arabidopsis transcription factor,DREB2A, involved in drought-responsive gene expression. Plant Cell 18:1292–1309.

10. Liu Q, et al. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. PlantCell 10:1391–1406.

11. Tang N, Zhang H, Li X, Xiao J, Xiong L (2012) Constitutive activation of transcriptionfactor OsbZIP46 improves drought tolerance in rice. Plant Physiol 158:1755–1768.

12. Aguirrezabal L, et al. (2006) Plasticity to soil water deficit in Arabidopsis thaliana:Dissection of leaf development into underlying growth dynamic and cellular variablesreveals invisible phenotypes. Plant Cell Environ 29:2216–2227.

13. Peres A, et al. (2007) Novel plant-specific cyclin-dependent kinase inhibitors inducedby biotic and abiotic stresses. J Biol Chem 282:25588–25596.

14. Cosgrove DJ (1997) Assembly and enlargement of the primary cell wall in plants.Annu Rev Cell Dev Biol 13:171–201.

15. Vriezen WH, Zhou Z, Van Der Straeten D (2003) Regulation of submergence-inducedenhanced shoot elongation in Oryza sativa L. Ann Bot (Lond) 91(Spec No):263–270.

16. Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B to its nuclear sig-nalling partner PIF3 is reversibly induced by light. Nature 400:781–784.

17. Shimizu-Sato S, Huq E, Tepperman JM, Quail PH (2002) A light-switchable genepromoter system. Nat Biotechnol 20:1041–1044.

18. Leivar P, Quail PH (2011) PIFs: Pivotal components in a cellular signaling hub. TrendsPlant Sci 16:19–28.

19. Maruyama K, et al. (2012) Identification of cis-acting promoter elements in cold- anddehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean. DNARes 19:37–49.

20. Hiratsu K, Matsui K, Koyama T, Ohme-Takagi M (2003) Dominant repression of targetgenes by chimeric repressors that include the EAR motif, a repression domain, inArabidopsis. Plant J 34:733–739.

21. Nakashima K, et al. (2007) Functional analysis of a NAC-type transcription factorOsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J51:617–630.

22. Matsukura S, et al. (2010) Comprehensive analysis of rice DREB2-type genes thatencode transcription factors involved in the expression of abiotic stress-responsivegenes. Mol Genet Genomics 283:185–196.

23. Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix tran-scription factor family. Plant Cell 15:1749–1770.

24. Dubouzet JG, et al. (2003) OsDREB genes in rice, Oryza sativa L., encode transcriptionactivators that function in drought-, high-salt- and cold-responsive gene expression.Plant J 33:751–763.

25. Khanna R, et al. (2004) A novel molecular recognition motif necessary for targetingphotoactivated phytochrome signaling to specific basic helix-loop-helix transcriptionfactors. Plant Cell 16:3033–3044.

26. Franklin KA, et al. (2011) Phytochrome-interacting factor 4 (PIF4) regulates auxinbiosynthesis at high temperature. Proc Natl Acad Sci USA 108:20231–20235.

27. de Lucas M, et al. (2008) A molecular framework for light and gibberellin control ofcell elongation. Nature 451:480–484.

28. Magneschi L, Kudahettige RL, Alpi A, Perata P (2009) Expansin gene expression andanoxic coleoptile elongation in rice cultivars. J Plant Physiol 166:1576–1580.

29. Choi D, Lee Y, Cho HT, Kende H (2003) Regulation of expansin gene expression affectsgrowth and development in transgenic rice plants. Plant Cell 15:1386–1398.

30. Sauter M, Mekhedov SL, Kende H (1995) Gibberellin promotes histone H1 kinaseactivity and the expression of cdc2 and cyclin genes during the induction of rapidgrowth in deepwater rice internodes. Plant J 7:623–632.

31. Cosgrove DJ, Bedinger P, Durachko DM (1997) Group I allergens of grass pollen as cellwall-loosening agents. Proc Natl Acad Sci USA 94:6559–6564.

32. McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins thatinduce cell wall extension in plants. Plant Cell 4:1425–1433.

33. McQueen-Mason S, Cosgrove DJ (1994) Disruption of hydrogen bonding betweenplant cell wall polymers by proteins that induce wall extension. Proc Natl Acad Sci USA91:6574–6578.

34. Shcherban TY, et al. (1995) Molecular cloning and sequence analysis of expansins—Ahighly conserved, multigene family of proteins that mediate cell wall extension inplants. Proc Natl Acad Sci USA 92:9245–9249.

35. Lamport DT, Kieliszewski MJ, Chen Y, Cannon MC (2011) Role of the extensin su-perfamily in primary cell wall architecture. Plant Physiol 156:11–19.

36. Cannon MC, et al. (2008) Self-assembly of the plant cell wall requires an extensinscaffold. Proc Natl Acad Sci USA 105:2226–2231.

37. Li M, et al. (2009) Rice cellulose synthase-like D4 is essential for normal cell-wallbiosynthesis and plant growth. Plant J 60:1055–1069.

38. Nakamura Y, Kato T, Yamashino T, Murakami M, Mizuno T (2007) Characterization ofa set of phytochrome-interacting factor-like bHLH proteins in Oryza sativa. BiosciBiotechnol Biochem 71:1183–1191.

39. Ito Y, et al. (2006) Functional analysis of rice DREB1/CBF-type transcription factorsinvolved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153.

40. Xiang Y, Tang N, Du H, Ye H, Xiong L (2008) Characterization of OsbZIP23 as a keyplayer of the basic leucine zipper transcription factor family for conferring abscisicacid sensitivity and salinity and drought tolerance in rice. Plant Physiol 148:1938–1952.

41. Nozue K, et al. (2007) Rhythmic growth explained by coincidence between internaland external cues. Nature 448:358–361.

42. Huq E, Quail PH (2002) PIF4, a phytochrome-interacting bHLH factor, functions asa negative regulator of phytochrome B signaling in Arabidopsis. EMBO J 21:2441–2450.

43. Lorrain S, Allen T, Duek PD, Whitelam GC, Fankhauser C (2008) Phytochrome-medi-ated inhibition of shade avoidance involves degradation of growth-promoting bHLHtranscription factors. Plant J 53:312–323.

44. Kunihiro A, et al. (2011) Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) ac-tivate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidencemechanism underlying photoperiodic control of plant growth of Arabidopsis thali-ana. Plant Cell Physiol 52:1315–1329.

45. Casson SA, et al. (2009) Phytochrome B and PIF4 regulate stomatal development inresponse to light quantity. Curr Biol 19:229–234.

46. Kumar SV, et al. (2012) Transcription factor PIF4 controls the thermosensory activa-tion of flowering. Nature 484:242–245.

47. Koini MA, et al. (2009) High temperature-mediated adaptations in plant architecturerequire the bHLH transcription factor PIF4. Curr Biol 19:408–413.

48. Potters G, Pasternak TP, Guisez Y, Palme KJ, Jansen MA (2007) Stress-induced mor-phogenic responses: Growing out of trouble? Trends Plant Sci 12:98–105.

49. Barnes PW, Flint SD, Caldwell MM (1990) Morphological responses of crop and weedspecies of different growth forms to ultraviolet-B radiation. Am J Bot 77:1354–1360.

50. Takasaki H, et al. (2010) The abiotic stress-responsive NAC-type transcription factorOsNAC5 regulates stress-inducible genes and stress tolerance in rice. Mol Genet Ge-nomics 284:173–183.

51. Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure,thermal perturbation of expression and transcript splicing, and promoter activityfollowing transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689.

52. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F (1999) Iron fortification of riceseed by the soybean ferritin gene. Nat Biotechnol 17:282–286.

53. Mitsuda N, et al. (2006) Efficient production of male and female sterile plants byexpression of a chimeric repressor in Arabidopsis and rice. Plant Biotechnol J 4:325–332.

54. Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencingusing siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13.

55. Kojima M, et al. (2009) Highly sensitive and high-throughput analysis of plant hor-mones using MS-probe modification and liquid chromatography-tandem mass spec-trometry: An application for hormone profiling in Oryza sativa. Plant Cell Physiol 50:1201–1214.

15952 | www.pnas.org/cgi/doi/10.1073/pnas.1207324109 Todaka et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 18

, 202

0