Rice OsIAA6 interacts with OsARF1 and regulates leaf inclination

Meiqing Xing,Wei Wang,Xing Fang,Hongwei Xuea,,*

a Shanghai Collaborative Innovation Center of Agri-Seeds,Joint Center for Single Cell Biology,School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China

b National Key Laboratory of Plant Molecular Genetics,CAS Center for Excellence in Molecular Plant Sciences,Chinese Academy of Sciences,Shanghai 200032,China

Keywords:Rice OsARF1 OsIAA6 Leaf inclination Auxin Brassinosteroids

ABSTRACT Leaf inclination,a component of crop architecture,influences photosynthetic efficiency and planting density.Various factors,particularly the phytohormones auxin and brassinosteroids(BRs),function in regulating lamina joint bending,and understanding of the genetic control of leaf inclination will help to elucidate the relevant regulatory network.Screening a rice T-DNA insertion population revealed a mutant that was insensitive to auxin and displayed an enlarged leaf angle due to increased cell length on the adaxial side of the lamina joint.Genetic analysis revealed that the increased leaf inclination was caused by T-DNA insertion in the promoter region of OsIAA6,resulting in elevated OsIAA6 expression.Further study showed that OsIAA6 interacts with OsARF1 to suppress auxin signaling and regulates leaf inclination.OsIAA6 mediates the BR effects on lamina joint development,and OsBZR1,the key transcription factor in BR signaling,binds directly to the promoter of OsIAA6 to stimulate its transcription.These results indicate the roles of the OsIAA6-OsARF1 module in regulating rice leaf inclination and suggest the synergistic effects of the phytohormones auxin and BR.

1.Introduction

Rice(Oryza sativa)is a model species for monocotyledonous plants and a staple human food.Improvement of crop architecture is an effective approach to increasing yield and has been a focus of cereal crop studies[1,2].Leaf inclination(the angle between the leaf blade and the culm)is a component of crop architecture.Erect leaves have a higher leaf area index and increased photosynthetic efficiency,favoring the dense planting of rice and maize[2-4].

Leaf inclination of rice results mainly from the asymmetric cell division and elongation of adaxial and abaxial cells at the lamina joint[5,6].A recent systematic morphological and cytological study[6]revealed that the lamina joint development is a dynamic process and indicated that cell proliferation and expansion,cell wall thickening,and programmed cell death at the adaxial or abaxial sides form the cytological basis of the lamina joint.A complex regulatory network has been shown to participate in leaf angle regulation,among whose components are phytohormones and transcription factors[7].

A series of mutants exhibiting altered leaf angles are associated with the biosynthesis or signaling of phytohormones known as brassinosteroids(BRs)[8].Deficiency mutants of BR-biosynthesis genes including d11,osdwarf4,d2,brd1,and brd2 display upright leaves owing to the inhibited elongation of parenchyma cells at the adaxial side of the lamina joint[2,9-12].A deficiency of BR signaling components also leads to erect leaves.Transgenic plants with suppressed expression of BRASSINOSTEROID UPREGULATED 1(BU1)[13],INCREASED LEAF INCLINATION 1(ILI1)[14],BRASSINOSTEROID INSENSITIVE 1(OsBRI1)[15],DWARF AND LOW-TILLERING(DLT)[16],BRI1-ASSOCIATED KINASE 1(OsBAK1)[17],and BRASSINAZOLE-RESISTANT 1(OsBZR1)[18,19]show reduced leaf inclination.The APETALA2 transcription factor REDUCED LEAF ANGLE 1/SMALL ORGAN SIZE 1(RLA1/SMOS1)interacts with and is phosphorylated by GLYCOGEN SYNTHASE KINASE3-LIKE GENE 2(OsGSK2),and deficiency of RLA1/SMOS1 results in upright leaves[20].LEAF INCLINATION 2(OsLC2)encodes a VERNALIZATIONINSENSITIVE 3-like protein and represses BR signaling and leaf inclination by controlling the expansion and division of adaxial cells of the lamina joint[7,21].

Besides BR,auxin,gibberellins and ethylene also play roles in leaf angle regulation[22].Auxin regulates leaf development and vascular differentiation[23]and as with BRs,altered biosynthesis or signaling of auxin lead to changes in leaf inclination.Auxin negatively regulates leaf angle,and rice plants with reduced auxin levels including the gain-of-function mutant lc1[24](OsGH3-1)or transgenic lines overexpressing GH3 family members OsGH3-2,OsGH3-5,and OsGH3-13,display increased leaf inclination[25-27].Similarly,decreased expression of the auxin receptors OsTIR1 or OsAFB2 results in increased leaf inclination[28-30].LEAF INCLINATION 4(LC4)encodes an F-box protein and is targeted by OsmiR394.The OsmiR394-LC4 module mediates the inhibitory auxin effects on leaf inclination[31].

Auxin triggers rapid transcriptional response,including of the auxin-responsive genes Aux/IAAs,ARFs,SAUR,and GH3s.Aux/IAAs interact with distinct ARFs to regulate the expression of downstream genes and thereby mediate the auxin effects on specific processes.OsIAA1 may interact with OsARF1 to regulate rice morphogenesis[29].In a recent study[32]LEAF INCLINATION 3(OsLC3),a SPOC domain-containing transcription suppressor,was shown to regulate OsIAA12 and OsGH3.2 expression via interaction with transcription factor LC3-interacting protein 1(LIP1).OsIAA12 also interacts with OsARF17 to increase leaf inclination(OsIAA12 overexpression or OsARF17 deficiency result in enlarged leaf inclination).

Most auxin-associated mutants with altered leaf angle show changed BR responses[24,26,29],suggesting crosstalk between BR and auxin in lamina joint development.OsARF19 regulated the expression of OsBRI1 and thereby leaf angle by binding the promoter region of OsBRI1[26].The mutant ds1 showed reduced BR sensitivity and leaf angle,and DS1 regulates rice architecture by interacting with OsARF11 to regulate OsBRI1 expression[33].

The mechanisms underlying the effects of BR and auxin on leaf inclination formation remain to be uncovered.To further elucidate the regulatory network of leaf inclination,we screened the rice TDNA insertion population and identified a gain-of-function mutant showing enlarged leaf angle.Genetic studies indicated that OsIAA6 interacts with OsARF1 to regulate leaf angle,and OsBZR1 stimulates directly the transcription of the OsIAA6 gene,providing informative clues to the synergetic effects of auxin and BR in regulating leaf inclination.

2.Materials and methods

2.1.Plant materials and growth conditions

Plants of cultivar Zhonghua 11(Oryza sativa,japonica,ZH11)were used as the wild type.An auxin-insensitive mutant arr1(iaa6-D)was identified in the Shanghai T-DNA Insertion Population(SHIP)[34].ZH11,the iaa6-D mutant,and various transgenic lines were grown in the greenhouse under a 12-h light(28°C)and 12-h dark(22 °C)regime.For phenotypic observation,rice plants were grown in an experimental paddy field in Shanghai under natural conditions.

For auxin treatment,seedlings of ZH11 and arr1(iaa6-D)were grown on 0.8% agar supplemented with exogenous indole-3-acetic acid(IAA,0,0.01,0.1,1,and 10μmol L-1)for 7 days,and the lengths of the seminal roots were recorded.The bioassay of rice lamina joint inclination was performed as previously described[35].

2.2.Identification of iaa6-D mutant

Thermal asymmetric interlaced(TAIL)PCR was applied to identify the flanking sequence of the insertion site in the arr1(iaa6-D)mutant.To confirm the T-DNA insertion,primers IAA6-F and IAA6-R,together with LB3(IAA6-F,5′-TACGCCACAACCAACAGCAG-3′;IAA6-R,5′-ATGATTGAGGCAGCCCCAGA-3′;LB3,5′-TAGCATCT GAATTTCATAACCAATCTCGATACAC-3′)were used.The presence of a single-copy T-DNA insertion in arr1(iaa6-D)was verified by statistics of segregation ratios.

2.3.Leaf angle measurement and cytological analysis

Leaf angles between sheath and leaf of ZH11 and arr1(iaa6-D)mutant at 10 days after heading were photographed and measured with ImageJ.The lamina joints from flag leaves of ZH11 and arr1(iaa6-D)were cut and fixed in FAA solution(37% formaldehyde:acetic acid:70% alcohol,5:5:90,v:v:v),dehydrated in a graded ethanol series,and embedded in Epon812 resin(Fluka,Heidelberg,Germany).Microtome sections(8μm)were cut and stained with toluidine blue.Sections were observed and photographed to determine the cell length and number of cell layers on the adaxial side.

2.4.RNA extraction and quantitative real-time PCR(qPCR)

Total RNAs were extracted with Trizol reagent(Invitrogen,Carlsbad,CA,USA)and cDNA was synthesized by reverse transcription(Takara,Kyoto,Japan).qPCR analysis was performed using SYBR PCR Master Mix(Toyobo,Osaka,Japan).Rice gene ACTIN was amplified for normalizing the expression levels of genes.Primers used are listed in Table S1.All experiments were biologically repeated three times.

2.5.Promoter-reporter fusion studies

A～2.5-kb DNA region of OsIAA6 promoter was amplified using primers IAA6-P1 and IAA6-P2.The amplified DNA fragment was subcloned into the pCAMBIA1300+pBI101.1 vector[36]to drive theβ-glucuronidase(GUS)reporter gene.The confirmed construct was transformed into ZH11 by Agrobacterium tumefaciens(strain EHA105)-mediated transformation using immature embryo[37].Positive transgenic lines were used for GUS staining[38]and observed.

2.6.Yeast-two hybrid assays

Yeast two-hybrid assays and measurement ofα-galactosidase activity were performed according to the Yeast Protocols Handbook & Matchmaker user manual(Clontech,Mountain View,CA,USA).Full coding regions of rice IAAs and ARFs were amplified and subcloned into pGADT7 and pGBKT7 vectors respectively(Clontech).The confirmed constructs were co-transformed into yeast strain AH109 and yeast growth was observed after 4 days.Primers are listed in Table S2.

2.7.Split-luciferase assays

For split-luciferase assay,the coding sequence of rice IAA6 was subcloned into the nLUC vector,and those of rice ARF1,ARF6,ARF7,ARF12,and ARF16 were subcloned into the vector cLUC.Tobacco leaves were infiltrated with different combinations of constructs and luciferase(LUC)activity was measured after infiltration for 48 h using a low-light cooled CCD imaging apparatus.Primers are listed in Table S2.

2.8.Cis-element analysis and electrophoretic mobility shift assay(EMSA)

The Plant Cis-acting Regulatory DNA Elements resource(https://www.dna.affrc.go.jp/PLACE/)was used to search the～2.5 kb region of OsIAA6 promoter for the presence of BRRE element-CGTGT/CG and E-Box-CANNTG.

For recombinant expression of OsBZR1-His fusion protein,the coding region of OsBZR1 was amplified and subcloned into pET-32a(+)(Novagen,Darmstadt,Germany).Recombinant protein was expressed in Escherichia coli BL21 strain and purified using Ni-NTA resin(Qiagen,Hilden,Germany).

Fig.1.Rice auxin resistant of root growth 1,arr1,is insensitive to auxin and presents enlarged leaf inclination.(A)Rice arr1 is insensitive to exogenous auxin.Rice seedlings of WT(ZH11)and arr1 were grown on medium supplemented with exogenous IAA(0,0.01,0.1,1,and 10μmol L-1)for 7 days and observed(left,scale bar,2 cm).Root lengths were measured and calculated,and that of ZH11 without auxin treatment was set as‘‘1”.Values are means±SE(n=20)with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(B)qPCR analyses using leaves of 14-day-old seedlings revealed the reduced transcriptions of OsIAA1,OsIAA9,OsIAA19,and OsIAA24.Transcript levels of genes were normalized with ACTIN.The expressions of corresponding genes in ZH11 were set as 1.0.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(C)arr1 presents enlarged leaf inclination at heading stage(left,scale bar,20 cm;enlarged leaf angles are highlighted in middle,scale bar,2 cm).Leaf angles of ZH11 and arr1 plants at 10 days after heading were measured and compared by Student’s t-test(**,P＜0.01).Values are means±SE(n＞10)with three biological repeats.(D)Longitudinal sections through lamina joint of flag leaves of ZH11 and arr1 at 10 days after heading(left).Cell length and number of cell layers at the adaxial side were measured and compared by Student’s t-test(**,P＜0.01).

DNA fragments of the OsIAA6 promoter region containing CGTGT/CG and mutated version were PCR-amplified and labeled with a Digoxigenin PCR Labeling Kit(Roche,Mannheim,Germany).EMSA and competition experiments were performed as previously described[39].Primers used are listed in Table S3.

2.9.Luciferase activity assays

To investigate the transactivation by OsBZR1 of OsIAA6,the coding region of OsBZR1 was amplified and subcloned into pAN580(no GFP protein)as the effector construct.The～2.5-kb OsIAA6 promoter or minimal 35S promoter were subcloned separately into pGreenII-0800-LUC to generate reporter constructs.Effector and reporter constructs were co-transformed into Arabidopsis protoplasts.Preparation,transfection,and culture of Arabidopsis protoplasts were performed as previously described[40]and luciferase activity was measured as previously described[41].Primers used are listed in Table S3.

3.Results

3.1.Identification of arr1/iaa6-D mutant

Mutants with altered response to auxin were identified using SHIP.By observation of the growth of primary roots,auxin resistant of root growth 1(arr1)mutant was identified.Compared with the wild type ZH11,arr1 is insensitive to auxin with respect to its seminal root growth(Fig.1A).qPCR analysis showed that transcription of genes involving in auxin signaling including OsIAA1,OsIAA9,OsIAA19 and OsIAA24,which were dramatically upregulated by 2,4-Dichlorophenoxyacetic acid(2,4-D)treatment[42],were significantly reduced in arr1(Fig.1B),confirming the suppressed auxin response of arr1.

The arr1 mutant presented enlarged leaf angles compared with ZH11.Leaf inclination measurement of flag leaves at 10 days after heading revealed a significant enlargement of arr1(Fig.1C).Cosegregation was determined from the phenotype of T2progeny from the heterozygous arr1 mutant.All the mutants with T-DNA insertion showed the exaggerated leaf inclination,whereas plants without T-DNA insertion showed the wild-type phenotype,confirming the genetic linkage of arr1 with leaf inclination.

To characterize the cellular alteration of arr1 at the lamina joint,longitudinal sections were made.Cells on the adaxial side of arr1 collar were found to be twice as long as those of ZH11,whereas there was no alteration of cell layers(Fig.1D),indicating that the enlarged leaf angle of arr1 was due to the increased length of adaxial cells at the lamina joint.

The Basic Local Alignment Search Tool(BLAST)finds the T-DNA was located 436 bp upstream region of gene LOC_Os01g53880(https://rice.uga.edu/cgi-bin/ORF_infopage.cgi?orf=LOC_Os01

g53880.1),which encodes a rice IAA6 protein(Fig.2A).Analysis by PCR confirmed the T-DNA insertion(Fig.2A,bottom left)and qPCR analysis revealed the increased expression of OsIAA6 in the homozygous arr1 mutant(Fig.2A,bottom right),indicating that arr1 was a gain-of-function mutant.We designated arr1 as iaa6-D.

To demonstrate the effects of OsIAA6 on leaf inclination,iaa6-D plants were transformed with OsIAA6-RNAi to repress the increased OsIAA6 expression.Observation of confirmed transgenic plants with recovered OsIAA6 expression(Fig.2B,left,6 independent homozygous lines were obtained)revealed normal leaf inclination(Fig.2B),showing the role of OsIAA6 in regulating leaf inclination.Transgenic ZH11 plants with increased expression of OsIAA6(driven by the OsIAA6 native promoter,Fig.2C,3 independent homozygous lines were obtained)displayed increased leaf inclination(Fig.2D),further confirming the effect of OsIAA6 in regulating rice leaf inclination.

Fig.2.ARR1 encodes OsIAA6.(A)Schematic of ARR1/OsIAA6 gene.Exons(filled boxes),introns(lines),UTR(empty boxes),T-DNA insertion(triangle)and used primers are indicated(upper).Primers P1 and P2,together with LB3,were used to confirm the position of a T-DNA insertion in arr1/iaa6-D and to identify homozygous plants by PCR amplification using genomic DNA as template(bottom left).qPCR analysis revealed the increased transcription of OsIAA6 in leaves of arr1/iaa6-D(bottom right).Transcription levels were normalized to that of ACTIN and the OsIAA6 expression in ZH11 was defined as 1.0.Values are means±SE with three biological repeats.(B)iaa6-D mutant with rescued expression of OsIAA6(pUbi:OsIAA6-RNAi in iaa6-D,left)presented normal leaf inclination(middle,scale bar,20 cm).Leaf angles at 10 days after heading are described in the right panel.Transcription level was normalized with that of ACTIN and the OsIAA6 expression in ZH11 was defined as 1.0.Values are means±SE(n＞10)with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(C)qPCR analysis showed increased expression of OsIAA6 in transgenic rice seedlings overexpressing OsIAA6 driven by the native promoter(pIAA6:OsIAA6 in ZH11).Transcription levels were normalized with that of ACTIN and the OsIAA6 expression in ZH11 was defined as 1.0.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(D)Phenotypic observation(left,scale bar,20 cm)and calculation(right)revealed the increased leaf inclination under OsIAA6 overexpression.Angle of flag leaves at 10 days after heading was calculated and values are presented as means±SE(n＞10)with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01,compared to ZH11).

3.2.OsIAA6 is expressed in lamina joint

OsIAA6 was expressed in various tissues including roots,leaves,spikes and seeds,with highest expression in leaves(Fig.3A).Promoter-GUS fusion study further indicated that OsIAA6 was highly transcribed in seedlings,roots,leaves and flowers,while relatively weakly expressed in shoots(Fig.3B).RNA in situ hybridization analysis further revealed the transcripts of OsIAA6 in the lamina joint(Fig.S1A),and global transcriptome analysis of rice IAA genes at lamina joints showed a distinct expression pattern of OsIAA6:increased expression during lamina joint development of the last three upper leaves(Fig.S1B).The high expression of OsIAA6 in lamina joints is in accord with the enlarged leaf angle of iaa6-D.

qPCR analysis showed that OsIAA6 is induced by auxin(Fig.S1C),a finding consistent with the insensitive response of iaa6-D to auxin.

3.3.OsIAA6 interacts with OsARF1

To identify the rice ARF protein that interacts with OsIAA6,a yeast two-hybrid assay was performed to investigate IAA-ARF interactions in rice.Preliminary results revealed a complex rice IAA-ARF interaction network(Fig.S2).Candidate interacting ARFs(OsARF1,OsARF6,OsARF7,OsARF12,and OsARF16)of OsIAA6 were further examined by split-luciferase assay through expressing Nterminal luciferase-fused OsIAA6(OsIAA6-nLuc)and C-terminal luciferase-fused ARFs(cLuc-OsARFs)in tobacco leaf epidermal.Results confirmed the interaction between OsIAA6 and OsARF1 or OsARF16(Fig.3C).

Fig.3.OsIAA6 is transcribed in various tissues and interacts with OsARFs.(A)qPCR analysis revealed the transcripts of OsIAA6 in various tissues,with relatively high expression in leaf.Transcription levels were normalized with that of ACTIN.Values are means±SE with three biological repeats.(B)Promoter-reporter gene(GUS)fusion studies revealed OsIAA6 expression in young seedlings at several stages(1-5),root(3-5),leaf(6),flower(7)and glume(8).Three independent confirmed transgenic lines were observed and representative images are shown.Scale bar,0.5 cm.(C)Split-luciferase assay confirmed the in vivo interaction of OsIAA6 and various OsARFs in N.benthamiana leaves.Coding regions of OsIAA6 and OsARFs were fused into pCAMBIA1300-nLuc and pCAMBIA1300-cLuc,respectively,and used for leaf dot infiltration.Agrobacterium strains expressing OsIAA6-nLuc and cLuc-SGT1 or cLuc-OsARF and nLuc-RAR were used as negative controls;strains expressing cLuc-SGT1 and nLuc-RAR were used as positive controls.(D)qPCR analysis of iaa6-D or iaa6-D plants expressing rice OsARF1(pUbi:OsARF1).OsARF1 expression levels were normalized with that of ACTIN and the OsARF1 expression in iaa6-D was defined as 1.0.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(E)Phenotypic observation showed that OsARF1 overexpression partially restored the enlarged leaf inclination of iaa6-D(Scale bar,20 cm).Leaf angles at 10 days after heading were calculated and values are means±SE(n＞10)with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).

To confirm the role of OsIAA6-OsARF1 or OsIAA6-OsARF16 interaction in leaf angle regulation,OsARF1 or OsARF16 was overexpressed in iaa6-D driven by a constitutive ubiquitin promoter(pUbi:OsARF1 in iaa6-D).Transgenic lines were confirmed(Fig.3D)and analysis revealed that increased OsARF1 expression resulted in the normal leaf angle of iaa6-D(Fig.3E),while OsARF16 overexpression could not rescue the enlarged leaf angles of iaa6-D,indicating that OsIAA6 regulates rice leaf inclination by suppression of OsARF1 function.

3.4.iaa6-D is hypersensitive to BR

In addition to IAA,OsIAA6 expression is induced by BL(Fig.S1C).Considering the important role of BR in regulating leaf angle,we investigated the sensitivity of iaa6-D to BR.Observation of primary root length showed that iaa6-D is hypersensitive to BR(Fig.4A).Further examination of lamina bending under different concentrations of 24-epibrassinoide(24-eBL)showed that the lamina bending of iaa6-D is more marked than that of ZH11(Fig.4B),suggesting that OsIAA6 overexpression results in an increased response to BR.Examination of the transcription levels of BRbiosynthesis-related genes indeed showed decreased expression of DWARF4 and CPD1 in iaa6-D(Fig.4C),confirming the increased BR signaling of iaa6-D and suggesting that OsIAA6 might mediate BR regulation of leaf inclination.

3.5.OsBZR1 binds directly to OsIAA6 promoter

BR signaling is perceived by a receptor-like kinase,BRI1,which triggers the phosphorylation of downstream signaling components and finally activates the transcription factors BZR1 and BES1[43,44].In Arabidopsis,genome-wide analysis by chromatin immunoprecipitation microarray identified various target genes of BZR1 and BES1 and many of them were involved in auxin signaling pathway[44,45].BZR1 can bind directly to the promoter regions of both IAA19 and ARF7 to regulate the differential growth of Arabidopsis hypocotyls[39].Indeed,repression of OsBZR1 results in a reduced,and its overexpression in an enlarged,leaf angle in rice[19].

Previous studies in Arabidopsis showed that BRRE(CGTGT/CG)is the optimal binding site for BZR1 and BZR1 directly binds to BRRE to regulate the transcription of target genes[46].Two BRREs were identified in the OsIAA6 promoter region(Fig.5A,upper),suggesting that OsBZR1 might bind directly to the promoter and regulate the expression of OsIAA6.EMSA was performed using two fragments of the OsIAA6 promoter(probe 1,-1031 to-1170 bp;probe 2,-2331 to-2456 bp)including BRRE.OsBZR1 bound directly to probe 2(Fig.5A,bottom),whereas no binding was observed when the BRRE was mutated(Fig.5B),confirming that OsBZR1 binds specifically to the OsIAA6 promoter.

Fig.4.Altered BR response of iaa6-D.(A)iaa6-D displayed increased responses to exogenous 24-eBL.Rice seeds were soaked in water for two days in the dark,after which seeds with synchronous germination were transferred to and grown on medium supplemented with 24-eBL(0,0.01,0.1,and 1μmol L-1)for 7 days.Primary root growth was observed(upper)and recorded(bottom).Values are means±SE(n=20)and significant difference was determined by Student’s t-test(**,P＜0.01).(B)iaa6-D mutant showed increased inclination of the etiolated leaf lamina joint under 24-eBL treatment.After culture in darkness for 8 days,the second leaf joints of ZH11 and iaa6-D were floated in distilled water containing dosed concentrations of 24-eBL(0,0.01,and 0.1μmol L-1)for 48 h(upper).Leaf angle was measured by protractor and presented as means±SE(n＞15,bottom).(C)qPCR analysis revealed the decreased expression of BR-biosynthesis-related genes OsDWARF4 and OsCPD in iaa6-D plants.Transcript levels of examined genes were normalized with that of ACTIN and the expression of examined genes in ZH11 was defined as 1.0.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).

In a further investigation of the effect of OsBZR1 on OsIAA6 transcription,a luciferase activity assay by transient expression in Arabidopsis protoplasts showed that OsBZR1 binds to the promoter and stimulates the expression of OsIAA6 in vivo(Fig.5C).Indeed,qPCR analysis revealed the increased OsIAA6 transcription in rice plants overexpressing OsBZR1(Fig.5D),indicating that OsBZR1 stimulates OsIAA6 expression via direct binding to the promoter.

4.Discussion

4.1.OsIAA6 regulates leaf inclination via interaction with OsARF1

Although auxin synthesis and signaling are involved in leaf angle regulation[7],the underlying molecular mechanisms,in particular the distinct Aux/IAA-ARF proteins,are unknown.We have shown that OsIAA6,an Aux/IAA member reported[47]to be involved in drought tolerance and tiller growth,regulates leaf angle via interaction with OsARF1.Increased OsIAA6 expression suppresses auxin signaling and thereby promotes cell elongation at the adaxial side of the lamina joint,leading to enlarged leaf inclination(Fig.5E).Aux/IAA proteins interact with ARFs to suppress auxin signaling[48].Overexpression of OsARF1 results in the recovered leaf inclination of iaa6-D,while that of OsARF16 does not,suggesting that OsIAA6 may be involved in other developmental processes through interaction with OsARF16.

Fig.5.OsIAA6 is directly regulated by OsBZR1.(A)Rice BZR1 binds directly to the promoter region of OsIAA6.Positions of BR response element(BRRE,red)of OsIAA6 promoter region,probe 1(-1170 to-1031 bp)and probe 2(-2456 to-2331 bp)are highlighted(upper).EMSA showed that purified OsBZR1 protein bound to probe 2(bottom).Unlabeled DNA fragments(2-,5-,10-,and 20-fold in concentration)were used as competitors.(B)EMSA confirmed the binding of OsBZR1 to the OsIAA6 promoter region containing a CGTGTG motif.The same DNA fragment lacking the CGTGTG motif(Δprobe)was examined.(C)A transient expression assay showed that OsBZR1 bound to the OsIAA6 promoter to promote OsIAA6 expression in vivo.Effector(p35S:OsBZR1)and reporter(p35S:LUC or pIAA6:LUC)were transiently expressed in Arabidopsis protoplasts and luciferase activity was measured.The activity of p35S:LUC was defined as 1.0.Minimal 35S promoter and～2.5-kb OsIAA6 promoter were used.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01).(D)qPCR analysis revealed increased OsIAA6 expression in plants overexpressing OsBZR1.Fourteen-day-old seedlings of Nipponbare(wild type,WT)and transgenic plants overexpressing OsBZR1(OsBZR1-OE)were used for examination.OsIAA6 expression level was normalized with that of UBQ5 and relative expressions were calculated by defining the OsIAA6 expression in WT as 1.0.Values are means±SE with three biological repeats.Significant difference was determined by Student’s t-test(**,P＜0.01,compared to WT).(E)A hypothetical model illustrating OsIAA6 function in regulating leaf inclination.OsIAA6 interacts with OsARF1 to suppress auxin signaling and to promote cell elongation at the adaxial side of the lamina joint,leading to enlarged leaf inclination.OsBZR1 binds directly to OsIAA6 promoter and OsIAA6 mediates the synergistic effects of BR and auxin on lamina joint development.OsIAA6 may interact with other OsARFs to regulate other developmental processes.

In Arabidopsis,loss-of-function mutants of a few Aux/IAAs present subtle or indiscernible phenotypes[39,49,50],owing mainly to the functional redundancy of the Aux/IAA family.Similarly,the rice T-DNA insertion mutant OsIAA1 showed no phenotype under normal growth or auxin treatment[29].Indeed,suppressed OsIAA6 expression did not result in altered leaf inclination(Fig.S3),again suggesting the functional redundancy of rice Aux/IAAs.

Our previous study[32]indicated that OsIAA12 interacts with OsARF17 to regulate rice lamina joint development and that OsIAA12 overexpression resulted in enlarged leaf inclination.Identification of OsIAA6-OsARF1 provides the function of another pair of distinct rice IAA-ARF,in addition to the OsIAA12-OsARF17 module,in regulating leaf inclination.Whether OsIAA6 and other members of Aux/IAA proteins are coordinated in lamina joint development and leaf inclination regulation awaits investigation.

4.2.OsIAA6 mediates auxin-BR crosstalk in leaf inclination

BRs are key regulators of leaf angle in rice and maize[2,4,20,51]and most mutants with altered auxin synthesis or signaling show changed BR sensitivity.Previous studies[52,53]showed that the auxin response element(AuxRE)is a crosstalk point for auxin and BR signaling.Rice OsARF11 and OsARF19 bind directly to the AuxRE of the OsBRI1 promoter[5,26]and we have shown here that the BR response element(BRRE)also acts as a crosstalk point for BR and auxin crosstalk.OsBZR1 binds to the BRRE of the OsIAA6 promoter,indicating that OsIAA6 mediates the BR effect on leaf inclination regulation,similarly to the manner in which Arabidopsis BZR1 directly regulates the expression of IAA19 and ARF7 via binding to their promoter region[39].

Thus,OsIAA6 promotes leaf inclination via two pathways,the OsIAA6-OsARF1 module by interaction and OsBZR1-OsIAA6 by mediating BR-auxin crosstalk(Fig.5E),identifying a novel factor of leaf inclination regulation and a network in which auxin and BR synergistically determine leaf angle.

4.3.Aux/IAA and ARF interaction network

Plants respond rapidly to auxin signaling,and Aux/IAA transcriptional repressors suppress auxin signaling via interaction with ARF proteins[48,54,55].Based on large-scale analysis,an interactome map of Arabidopsis Aux/IAA proteins has been developed[55].In rice,there are 31 Aux/IAA proteins and 25 ARF proteins,and interactions among some members have been reported[56,57].We accordingly performed yeast two-hybrid analysis to systematically investigate Aux/IAA-ARF interactions.The results showed the complex and diverse interaction between rice Aux/IAAs and ARFs(Fig.S2A),which is possibly due to the similar structure of Aux/IAA and ARF proteins that leads to functional redundancy[58].

The interaction and regulation of Aux/IAA and ARF proteins are associated with their structure.Domains III and IV of ARF proteins determine the formation of homo-or hetero-interactions.Compared to ARF proteins containing domains III and IV,five members of ARF proteins(OsARF2,3,14,15 and 20)absent domains III and IV showed less interaction with Aux/IAA proteins in our study.ARFs could act as transcriptional repressors or activators.In rice,ARF activators interact with many more Aux/IAA proteins than ARF repressors,a finding consistent with that in Arabidopsis[59].

CRediT authorship contribution statement

Meiqing Xing:Investigation,Data curation,Writing-original draft.Wei Wang:Investigation,Data curation.Xing Fang:Investigation,Data curation.Hongwei Xue:Funding acquisition,Supervision,Writing-review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Ten Thousand Talent Program.We thank Dr.Dongfeng Liu from Prof.Kang Chong’s laboratory(Institute of Botany,Chinese Academy of Sciences)for help with OsBZR1-overexpressing transgenic plants.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.02.010.