Rice Homeobox Protein KNAT7 Integrates the PathwaysRegulating Cell Expansion and Wall Stiffness1[OPEN]

Shaogan Wang,a,2 Hanlei Yang,a,b,2 Jiasong Mei,a,b Xiangling Liu,a Zhao Wen,a Lanjun Zhang,a

Zuopeng Xu,a Baocai Zhang,a,3 and Yihua Zhoua,b,3,4

aState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, The InnovativeAcademy of Seed Design, Chinese Academy of Sciences, Beijing 100101, ChinabUniversity of Chinese Academy of Sciences, Beijing 100049, China

ORCID IDs: 0000-0002-9841-6084 (S.W.); 0000-0003-1088-9424 (H.Y.); 0000-0002-9213-0659 (J.M.); 0000-0003-0202-0817 (X.L.);0000-0001-5464-6622 (Z.W.); 0000-0002-0580-5743 (L.Z.); 0000-0001-9052-6185 (Z.X.); 0000-0002-3239-7263 (B.Z.); 0000-0001-6644-610X (Y.Z.).

During growth, plant cells must coordinate cell expansion and cell wall reinforcement by integrating distinct regulatorypathways in concert with intrinsic and external cues. However, the mechanism underpinning this integration is unclear, asfew of the regulators that orchestrate cell expansion and wall strengthening have been identified. Here, we report a rice (Oryzasativa) Class II KNOX-like homeobox protein, KNOTTED ARABIDOPSIS THALIANA7 (KNAT7), that interacts with differentpartners to govern cell expansion and wall thickening. A loss-of-function mutation in KNAT7 enhanced wall mechanicalstrength and cell expansion, resulting in improved lodging resistance and grain size. Overexpression of KNAT7 gave rise tothe opposite phenotypes, with plants having weaker cell walls and smaller grains. Biochemical and gene expression analysesrevealed that rice KNAT7 interacts with a secondary wall key regulator, NAC31, and a cell growth master regulator, Growth-Regulating Factor 4 (GRF4). The KNAT7-NAC31 and KNAT7-GRF4 modules suppressed regulatory pathways of cellexpansion and wall reinforcement, as we show in internode and panicle development. These modules function insclerenchyma fiber cells and modulate fiber cell length and wall thickness. Hence, our study uncovers a mechanismfor the combined control of cell size and wall strengthening, providing a tool to improve lodging resistance and yield inrice production.

Plants have.40 cell types, each with a unique shapeand function that depends in part on its wall properties(Chebli and Geitmann, 2017). Plant cell walls are a rigidand plastic network of polysaccharides (cellulose, hemi-cellulose, and pectins), aromatic compounds (lignin) andglycoproteins that encase plant cells (Bacic et al., 1988;Carpita and Gibeaut, 1993). Cell wall biogenesis andremodeling are closely related to all cell behaviors.For example, pectin demethylesterification affects cell

differentiation and organ initiation (Peaucelle et al., 2011),and synthesis and integration of wall products at thedivision plane is an important step in cytokinesis (CutlerandEhrhardt, 2002;Mayer and Jürgens, 2004). Cell growthinvolves cell expansion and wall reinforcement. Turgorpressure-triggered cell expansion requires relaxation ofthe cell wall, which involves the activities of xyloglucanendotranslycosylases and expansins (McQueen-Masonand Cosgrove, 1994; Whitney et al., 2000; Chanliaudet al., 2004; Che et al., 2015). While the cell walls areexpanding, newly synthesized polysaccharides are in-tegrated into the walls to provide rigidity. Upon mat-uration, secondary wall components are deposited insome types of cells, such as sclerenchyma fiber cells andvessel elements, to confer mechanical strength.Plants have evolved complex mechanisms to inte-

grate distinct signals to ensure that wall properties arecompatible with cell functions (Somerville et al., 2004).Combinatorial controls at different scales are required.Manipulation of enzymatic activities during cell wallbiogenesis directly controls cell wall composition andorganization; spatiotemporal coregulation of cell wall-related gene expression represents another valid control,as cell wall chemistry is heterogeneous (Brown et al.,2005; Persson et al., 2005). During cell expansion, sev-eral kinds of transcription factors (TFs), including basichelix-loop-helix proteins, rice (Oryza sativa) Growth-Regulating Factor 4 (GRF4), APETALA2-type proteins,squamosa promoter binding-like proteins, and the

1This research was supported by the Ministry of Agriculture ofChina for Transgenic Research (2016ZX08009003-003), the NationalNature Science Foundation of China (91735303 and 31530051), theYouth Innovation Promotion Association, Chinese Academy of Sci-ences (2016094), and the State Key Laboratory of Plant Genomics.

2These authors contributed equally to the article.3Senior authors.4Author for contact: yhzhou@genetics.ac.cn.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Yihua Zhou (yhzhou@genetics.ac.cn).

Y.Z. and B.Z. conceived and supervised the study; S.W. and H.Y.conducted the major analyses in this work; J.M. prepared the con-struct of CRISPR/Cas9; X.L. performed gene transformations; Z.W.performed laser microdissection; L.Z. performed cell wall composi-tion analyses; Z.X. investigated agronomic traits; Y.Z. and S.W. wrotethe article.

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Myeloblastosis (MYB)-like TFs, form a regulatorynetwork (Aya et al., 2014; Che et al., 2015; Duan et al.,2015; Si et al., 2016; Jang et al., 2017; Wu et al., 2017).Wall stiffening is modulated by a regulatory networkconsisting of NAM, ATAF, and CUC (NAC) and MYBTFs (Zhong et al., 2010, 2011; Zhao, 2016). Moreover,cell wall biogenesis is affected by various develop-mental signals (Somerville et al., 2004), including planthormones, such as brassinosteroids, auxin, and gib-berellins, as well as light (Wang and He, 2004; Bai et al.,2012; Todaka et al., 2012; Aya et al., 2014; Shan et al.,2014; Huang et al., 2015). However, the mechanism thatintegrates different regulatory pathways to modulatecell expansion and wall strengthening remains elusive.

Knotted-related homeobox (KNOX) proteins may beinvolved in integrating these pathways, as their func-tions are related to diverse physiological processes(Hay and Tsiantis, 2010). KNOXmembers belong to theplant-specific three-amino acid loop extension super-class of homeodomain proteins (Bürglin, 1997; Hakeet al., 2004) and have been clustered into two classes(Kerstetter et al., 1994; Reiser et al., 2000; Magnani andHake, 2008). The functions of Class I members are im-plicated in many processes of the plant life cycle; one oftheir most important roles is to control cell proliferationat the leaf and shoot apical meristems (Vollbrecht et al.,1991; Long et al., 1996; Sato et al., 1999; Belles-Boix et al.,2006). Characterizations of the upstream regulators,interacting cofactors, and downstream effectors haveplaced Class I KNOX proteins in the central nodes invarious physiological processes (Hay and Tsiantis,2010).

Class II KNOX proteins are less well studied, buttheir widespread expression pattern indicates thattheir functions are as diverse as those of Class I(Truernit et al., 2006; Zhong et al., 2008; Chai et al.,2016). The most substantially studied Class II memberis KNOTTED ARABIDOPSIS THALIANA7 (KNAT7).It governs secondary wall formation either by inter-acting with secondary wall TFs, e.g. OVATE FAMILYPROTEIN4 and MYB75, or by being regulated by thesecondarywallmaster TFs, such as SECONDARYWALL-ASSOCIATED NAC DOMAIN PROTEIN/VASCULAR-RELATED NAC DOMAIN6 and MYB46 (Brown et al.,2005; Zhong et al., 2008; Ko et al., 2009; Bhargava et al.,2010; Li et al., 2011, 2012;Gong et al., 2014; Liu et al., 2014).However, the role of KNAT7 in integrating cell growthregulatory pathways is unknown.

Here, we report on the rice class II KNOX-like home-obox member KNAT7, which integrates the regulatorypathways of cell expansion and wall strengthening. Aloss-of-function mutation in KNAT7 resulted in en-hanced secondary wall biosynthesis and facilitatedcell expansion in the mutant; overexpression (OE) ofKNAT7 gave rise to the opposite effects. KNAT7 inter-acted with the secondary wall key regulator NAC31and the master cell growth factor GRF4 to repress theirdownstream regulatory pathways. These findings sug-gest that rice KNAT7 plays an integrative role in coor-dinating cell size andwall stiffness. Therefore, this study

provides insight into the combinatorial control of cellgrowth and may be instrumental in synergistically im-proving agronomic traits, especially grain size (and thusyield) and stem strength (and thus resistance of lodging),in crop breeding.

RESULTS

Rice KNAT7 Negatively Regulates Cell Wall Thickeningand Mechanical Properties

Cellulose contributes greatly to wall mechanical prop-erties (Cosgrove, 2005). To understand how rice plantsbuildwall rigidity, we performed coexpression analysiswith a characterized cellulose synthase gene, CESA4(Zhang et al., 2009), to screen for key regulators. Thisanalysis identified several NAC and MYB TFs and aClass II homeobox protein, KNAT7 (SupplementalTable S1). Phylogenetic analysis placed rice KNAT7as an ortholog of Arabidopsis (Arabidopsis thaliana)KNAT7 (Supplemental Fig. S1), but its function in riceis uncharacterized.

To identify the functions of rice KNAT7, we generateda mutant in the rice Zhonghua11 background using theclustered regularly interspaced short palindromicrepeats (CRISPR)/CRISPR associated protein9 (Cas9)gene editing approach (Supplemental Fig. S2A). Sequenceanalysis revealed a 19-bp deletion in the second exonof KNAT7, which causes a 48-bp reading-frame shiftat the 428-bp site of the coding region and results in apremature translational stop codon (Fig. 1, A and B).Compared to the predicted wild-type KNAT7 proteinsequence, the mutated KNAT7 is 137 amino acids inlength and lacks the conserved domains of KNOXproteins (Supplemental Fig. S1C). TheKNAT7OE lineswere produced by constitutively expressing KNAT7in the rice variety Nipponbare (Fig. 1C; SupplementalFig. S2B). In addition to the slightly reduced paniclelength and plant height (Supplemental Fig. S2C), theknat7 mutants had an increased mechanical strength,whereas the KNAT7-OE lines had compromised me-chanical force (Supplemental Fig. S2D), suggesting thatcell wall structure might be affected in these KNAT7-modulated plants. We therefore investigated the epi-dermal sclerenchyma fiber cells in the internodes of theseplants. Scanning electronmicroscopy (SEM) revealed thatthewall thickness of fiber cells was significantly increasedin the knat7 mutant but was decreased in the KNAT7-OElines when compared with the corresponding wild-typeplants (Fig. 1, D–F).

In agreementwith the anatomical alterations, cellulosecontent increased in the knat7 mutants and decreased inthe KNAT7-OE lines compared to the correspondingwild-type plants (Fig. 1G). Measurement of the neutralsugar content of cell wall residues showed that the Xylcontent, which represents the abundance of xylan, in-creased in the knat7mutant but slightly decreased in theKNAT7-OE lines compared to the corresponding wild-type plants (Supplemental Table S2).Moreover, the levelof lignin, another component of wall stiffness, was

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unchanged in the knat7mutant and slightly increased inthe OE plants when compared to the correspondingwild-type plants (Supplemental Table S2). Therefore,rice KNAT7 decreases secondary wall biosynthesis andmechanical strength.

Grain Size Is Altered in the knat7 Mutant and OE Plants

In addition to the changes in wall stiffness, manipu-lation of rice KNAT7 unexpectedly caused alterations ingrain size. The knat7mutant had larger grains, whereasthe KNAT7-OE lines had smaller grains than those ofthe corresponding wild-type plants (Fig. 2A). Quanti-tative measurements revealed that the increased grainsize of the knat7 mutant resulted from an increase ingrain length, grainwidth, and 1000-grainweight, whereasthe opposite phenotypeswere observed in theKNAT7-OElines (Fig. 2, B–D).To address the underlying cause of these changes

at the cellular level, we investigated the glumes ofwild-type, knat7, and KNAT7-OE grains using SEM.Compared to the corresponding wild-type plants, thecell length and cell width in the glumes were signif-icantly higher in the mutant plant and lower in theKNAT7-OE lines (Fig. 2, E–H), with slight changes inthe cell number (Fig. 2I). These findings suggest thatthe altered grain size in these KNAT7-modulated plants

is due to the variations in cell size. Hence, rice KNAT7plays a role in cell-size control in the glumes.

KNAT7 Affects Regulatory Pathways of Cell WallThickening and Cell Expansion

We next investigated the biochemical features of riceKNAT7. We transfected GFP-fused KNAT7 into leafepidermal cells of Nicotiana benthamiana and detectedGFP signals in the nucleus (Supplemental Fig. S3A),indicating that it is a putative TF.To explore whether rice KNAT7 has transactivation

activity, we coexpressed the effectors Pro-35S:GAL4BD(BD) or BD fusions to the herpes simplex virus VP16activationdomain (BD-VP16) and toKNAT7 (BD-KNAT7)with the reporter ProGAL4:Luciferase in Arabidopsisprotoplasts. In contrast to the strong expression ofluciferase promoted by BD-VP16, the reporter tran-scripts activated by BD-KNAT7 were low and similarto that promoted by BD (Supplemental Fig. S3B).Hence, KNAT7 was unable to activate the expressionof the reporter gene. We prepared another reporterconstruct ProKNOX-ProGAL4:Luciferase by placing fourcopies of the TGACmotif, the binding element of KNOXproteins, together with the GAL4 binding element. Thisconstruct provides binding elements for KNAT7 andBD-VP16. After cotransfecting Arabidopsis protoplasts

Figure 1. Rice KNAT7 represses wall thickening. A, Schema of KNAT7 gene structure and the mutation site of knat7. The boxesand lines in the diagram indicate exons and introns, respectively. The arrowhead indicates a 19-bp deletion in knat7, which resultsin a 48-bp reading-frame shift (underlined letters) and a premature translational stop codon (red letters). B, Genotyping the knat7plants using the primers (F1 R) shown in Supplemental Table S4 reveals the 19-bp deletion in the mutant. C, RT-qPCR analysis ofKNAT7 expression in theOE plants, showing the relative expression level of KNAT7 to riceHNR. Data represent themean6 SD ofthree biological replicates. Lowercase letters indicate significantly different means according to the variance analysis and Tukey’stest (P , 0.01). D and E, SEM graphs of sclerenchyma fiber cells from the internodes of the indicated plants. Bars 5 2 mm. F,Measurement of the wall thickness in sclerenchyma fiber cells of the indicated plants. Data represent means6 SD (n5 200 cellsfrom three individual internodes of the indicated plants). *P, 0.01 by Welch’s unpaired t test represents a significant differencefrom the correspondingwild-type plants. (G) Cellulose content in internodes of the indicated plants. Data represent themean6 SD

(n5 3 biological replicates). *P, 0.01 byWelch’s unpaired t test represents a significant difference from the corresponding wild-type plants. ZH11, Zhonghua11; NP, Nipponbare; L, line.

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with the reporter and effector combinations, the luciferaseactivities promoted by coexpressing BD-VP16 andKNAT7were significantly decreased compared to those activatedby expressing BD-VP16 andGUS (Supplemental Fig. S3C).Hence, rice KNAT7 can function as a transcriptionalrepressor.

To determine how rice KNAT7 controls both sec-ondary wall formation and cell expansion in planta,we investigated the transcription of genes implicatedin the regulatory pathways of secondary wall for-mation and cell expansion in the KNAT7 modulatedplants. NAC31-MYB61-CESAs is one of the identi-fied regulatory pathways for secondary wall cellu-lose synthesis in rice (Huang et al., 2015); MYB103is another crucial regulator for rice secondary wallthickening (Ye et al., 2015). Reverse transcription quan-titative PCR (RT-qPCR) analyses in the young inter-nodes revealed that the expression of those geneswas upregulated in the knat7 mutant but down-regulated in the KNAT7-OE lines (Fig. 3, A and C),suggesting that the regulatory pathways for secondarywall formation are affected in these KNAT7-modulatedplants.

Rice GRF4 is a major quantitative trait locus control-ling grain size, and expansin genes are its downstreameffectors (Che et al., 2015). While GRF4 transcript levelswere maintained at a similar level in the young spikeletsof the knat7 mutant and KNAT7-OE lines (Fig. 3B),transcription of the examined expansin genes wasupregulated in the knat7 mutant but suppressed inthe KNAT7-OE lines (Fig. 3D).

Therefore, KNAT7 controls the expression of genesimplicated in wall strengthening and cell expansion.

KNAT7 Interacts with NAC31 and GRF4 to Repress theDownstream Regulatory Pathways

The altered transcript levels discussed above promptedus to investigate whether KNAT7 can transcriptionallyregulate the examined genes. To this end, we transfectedArabidopsis protoplast cells with the effector constructPro-35S:KNAT7 and the reporter construct that harborsluciferase driven by the promoters of NAC31, MYB61,MYB103, and the expansin genes. Transactivation activityanalysis showed that KNAT7 cannot transcriptionally

Figure 2. Rice KNAT7 affects grain size. A, Rice grains of the indicated plants. Bar 5 5 mm. B to D, Measurement of the grainlength, grain width, and grain weight of the indicated plants. Data represent the mean value6 SD (n$ 15 grains harvested from atleast five plants), except in D, where data represent the mean value 6 SD (n 5 3 biological replicates). *P , 0.01 by Welch’sunpaired t test represents a significant difference from the corresponding wild-type plants. E and F, SEM graphs of the glume cellsof the indicated plants. Bars5 50 mm. G and H, Statistical analysis of the cell size in the glumes of the indicated plants based onthe SEM analyses. Data represent the mean6 SD (n$ 205 glume cells in grains from four plants). *P, 0.01 byWelch’s unpairedt test represents a significant difference from the corresponding wild-type plants. I, Statistical analysis of cell number per glume atthe longitudinal direction. Data indicate the mean value 6 SD (n $ 13 grains harvested from at least five plants). *P , 0.01 byWelch’s unpaired t test represents a significant difference from the corresponding wild-type plants. ZH11, Zhonghua11; NP,Nipponbare; L, line.

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regulate NAC31, MYB61, MYB103, or the expansingenes (Supplemental Fig. S4). Taken together withthe unchanged transcript levels of GRF4 in the trans-genic plants (Fig. 3B), KNAT7 might not act alone asa TF to modulate the downstream gene expressionin rice.Transactivation activity analysis further showed that

NAC31 transcriptionally activated itself in Arabidopsisprotoplasts expressing NAC31 and a reporter constructwith theNAC31promoter driving luciferase (SupplementalFig. S4A). Cotransfecting KNAT7 in these protoplastssuppressed the self-transactivation activity of NAC31(Supplemental Fig. S4A), implying that KNAT7 may

function through a protein interaction. To test thishypothesis, we performed several experiments thatdemonstrate protein-protein interactions. Split-luciferasecomplementation assays showed an interaction betweenKNAT7 and NAC31 (Fig. 4A), which was confirmedby yeast two hybrid analyses (Fig. 4B). In vivo bimo-lecular fluorescence complementation (BiFC) and single-molecule fluorescence resonance energy transfer (FRET)analyses revealed that the interactions occur in the nuclei(Fig. 4, C–E; Supplemental Fig. S5). Coimmunoprecipi-tation (co-IP) experiments in rice protoplasts providedadditional biochemical evidence for the interactions(Fig. 4F). Todetermine the interacting effects,we analyzedthe ability of NAC31 to activate MYB61 and MYB103transcription in the presence of KNAT7 in Arabidopsisprotoplasts. Transactivation activity assays demon-strated that the luciferase activities promoted byNAC31were significantly repressed by coexpression of KNAT7(Fig. 4G).We further determined the relationship between

KNAT7 and the cell growth regulator GRF4. Split-luciferase complementation and yeast two hybridassays revealed the interaction between KNAT7 andGRF4 (Fig. 5, A and B). The KNAT7-GRF4 complexwas found to form in the nuclei, based on the resultsof BiFC and single-molecule FRET analyses (Fig. 5, C–E;Supplemental Fig. S5). Co-IP assays in rice protoplastsbiochemically validated the interactions (Fig. 5F). Sim-ilarly, we performed transactivation activity analysesto examine the interaction effect. As shown in Figure 5G,KNAT7 suppressed the expression of the expansin genesEXPB3, EXPB17, and EXPA6 promoted by GRF4 in theprotoplasts.Moreover, the mutated KNAT7 protein that contains

the first 137 amino acids failed to interact with GRF4and NAC31 as revealed by split-luciferase comple-mentation analysis (Supplemental Fig. S6). Taken to-gether, these results suggest that KNAT7 interacts withkey regulators NAC31 and GRF4 to compromise theexpression of downstream genes.

KNAT7 Modules Function during Internode andPanicle Development

Next, we investigatedwhere the KNAT7-NAC31 andKNAT7-GRF4 modules function in planta. Severalorgans from Nipponbare were used to examine theexpression of KNAT7, NAC31, and GRF4. RT-qPCRanalyses showed that KNAT7 is ubiquitously expressedat relatively high levels in the internodes and panicles(Fig. 6A), in agreement with the tissues where the phe-notypes are displayed in the KNAT7-modulated plants.As developing internodes and panicles essentially pro-vide a time course of cell expansion and wall strength-ening (Itoh et al., 2005; Huang et al., 2015; Zhang et al.,2018), we explored the gene expression profile in thedeveloping internodes and panicles of wild-type plants.We prepared total RNA from eight segments of 9-cmwild-type internodes (Huang et al., 2015) and fromglumes

Figure 3. Gene expression analysis. A to D, RT-qPCR analysis of thetranscription of the examined genes involved in secondary wall for-mation and cell expansion in young internodes (A and C) and spikeletsfrom 10-cm panicles (B and D) of the KNAT7-modulated plants,showing the expression levels relative to the corresponding wild-typeplants. Rice TP1 was used for normalization of the expression of CESAgenes, and HNR was used for normalization of the expression of theother examined genes. Data represent the mean6 SD of three biologicalreplicates. *P, 0.01 by Welch’s unpaired t test represents a significantdifference from the corresponding wild-type plants. ZH11, Zhonghua11;NP, Nipponbare.

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of developing panicles 0.5 to 15 cm long. RT-qPCRanalyses in these tissues revealed that the expres-sion profiles of KNAT7 and NAC31 were similar inthe developing internodes (Fig. 6A), indicating thattheir translated proteins are able to encounter eachother during internode development. Specifically,KNAT7 was upregulated at segment 2 (sequentiallynumbered from the bottom up) and peaked at seg-ments 3 and 4, while NAC31 was upregulated atsegment 2 and decreased from segment 3 (Fig. 6A).Segments 2 and 3 of the internodes may be the stagesfor KNAT7-NAC31 interaction, consistent with thetiming of secondary wall initiation, as revealed by an-atomical analysis (Fig. 6B; Supplemental Fig. S7A).When these findings are combinedwith the phenotypesdisplayed in the internodes of the KNAT7-modulated

plants (Fig. 1), our observations validate the KNAT7-NAC31 module in internode development.

GRF4was mainly expressed in the panicles. Althoughthe transcript abundance ofGRF4 gradually declined asthe panicles matured, its transcript remained at a rela-tively high level (Fig. 6A). However, KNAT7 was upre-gulated in the 10-cm-long panicles and peaked in the15-cm panicles (Fig. 6A). The in planta interactionsbetween GRF4 and KNAT7 may occur in 10- to 15-cmpanicles. This claim was supported by our finding thatcell size in the glumes had 1-fold higher expansion in the10-cmpanicles than in the 5-cmpanicles, as shownby SEM(Fig. 6, C and D; Supplemental Fig. S7B). Taken togetherwith the alterations in cell size in the grains of KNAT7-modulated plants (Fig. 2), it is likely that the KNAT7-GRF4 module functions during panicle development.

Figure 4. Rice KNAT7 interacts with secondary wall regulator NAC31. A, Split-luciferase complementation assay showingthe interaction inN. benthamiana leaves infiltrated with the construct combinations shown at left. Rice GID1 was used as anegative control. Bar5 1 cm. B, Yeast two-hybrid analysis. Yeast cells were grown on SDmedium lacking Trp, Leu, His, andAde. Yeast growth status indicates interactions. C, BiFC analysis of the interaction in N. benthamiana leaves. Infiltrationswith the empty vector were used as negative controls (Supplemental Fig. S5). DAPI was used to visualize nuclei. Merge,merged images of enhanced YFP and DAPI. Bar 5 20 mm. D, FRET analysis in rice protoplasts verified the interaction. Thebottom row shows fluorescence in the cell after photobleaching YFP (AP). Bars 5 10 mm. E, Quantification of the FRETefficiency observed in D. FRET efficiency represents the fluorescence change of the donor fluorophore (cyan fluorescentprotein [CFP]) after photobleaching YFP. The background indicates the stability of CFP fluorescence before photobleaching.Rice GID1 protein was used as a negative control. Data represent the mean 6 SD (n 5 10 cells). *P , 0.01 by Welch’sunpaired t test. F, Co-IP analyses in rice protoplasts expressing FLAG-NAC31. GFP-GID1 was used as a negative control.G, Transcription activation was assayed by transfecting Arabidopsis protoplasts with the constructs shown at left. Datarepresent the mean 6 SD of three biological replicates. Lowercase letters indicate the different means according to thevariance analysis and Tukey’s test (P , 0.01).

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KNAT7 Represses the Regulatory Pathways of CellExpansion and Wall Strengthening

Given that the KNAT7-NAC31 and KNAT7-GRF4modules can form in the internodes and panicles,their effects need to be elucidated.MYB61 andMYB103are the downstream targets of NAC31, and theseMYB TFs target secondary wall CESA genes (Huanget al., 2015; Ye et al., 2015). RT-qPCR analyses in thewild-type internode segments showed that the tran-scription of MYB61, MYB103, and CESAs was sharplyupregulated at segment 2, peaked at segment 3, andgradually declined until maturation (Fig. 7A). The ex-pression pattern fits with the timing of the formationof the KNAT7-NAC31 module (Fig. 6A), indicatingthat the expression of secondary wall regulatory TFsand biosynthesis genes is quickly promoted by the

upstream TF genes, e.g. NAC31, in segment 2 andthen gradually repressed by the suppressors, e.g. theKNAT7-NAC31 module. Therefore, KNAT7-NAC31is one of the modules that can slow down wallthickening during internode development.Expansin genes are downstream targets of GRF4

(Che et al., 2015; Li et al., 2018). RT-qPCR analysesrevealed that the transcription of the four expansingenes was consistently upregulated in the 0.5- to10-cm panicles, peaked in the 10-cm panicles, anddropped to a low level in 15-cm panicles (Fig. 7B).The inflection points of these expression profilesmatch well with the feature of glume cell expansion(Fig. 6, C and D) and the timing to form the KNAT7-GRF4 module (Fig. 6A). Therefore, the KNAT7-GRF4module can suppress cell expansion during spikeletdevelopment.

Figure 5. Rice KNAT7 interacts with growth regulator GRF4. A, Split-luciferase complementation assay, showing the in-teraction in N. benthamiana leaves infiltrated with the construct combinations shown at left. Rice GID1 was used as anegative control. Bar 5 1 cm. B, Yeast two-hybrid analysis. Yeast cells were grown on SD medium lacking Trp, Leu, His,and Ade. Yeast growth status indicates interactions. C, BiFC analysis of the interaction between KNAT7 and GRF4 inN. benthamiana leaves. Infiltrations with the empty vector were used as negative controls (Supplemental Fig. S5). DAPI wasused to visualize nuclei. Merge, merged images of enhanced YFP and DAPI. Bar 5 20 mm. D, FRET analysis in rice protoplastsverifies the interaction. The bottom images show fluorescence in the cell after photobleaching YFP (AP). Bars 5 10 mm.E, Quantification of the FRET efficiency observed in D. FRET efficiency represents the fluorescence change of the donorfluorophore (CFP) after photobleaching YFP. The background indicates the stability of CFP fluorescence before photo-bleaching. Rice GID1 protein was used as a negative control. Data represent the mean 6 SD (n 5 10 cells). *P , 0.01 byWelch’s unpaired t test. F, Co-IP experiments in rice protoplasts expressing GFP-KNAT7. FLAG-GID1 was used as a negativecontrol. G, Transcription activation was assayed by transfecting Arabidopsis protoplasts with the constructs shown at left.Data represent the mean 6 SD of three biological replicates. Letters indicate the different means according to the varianceanalysis and Tukey’s test (P , 0.01).

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KNAT7 Coordinates Cell Expansion and WallStrengthening in Sclerenchyma Fiber Cells

We next addressed whether these KNAT7 mod-ules can act in a specific cell type. Sclerenchyma fibercells are one of the cell types affected by KNAT7, aswe observed the altered wall thickness in these cellsand the changed cellulose content in the internodes

of KNAT7-modulated plants (Fig. 1). Therefore, weexamined the size of sclerenchyma fiber cells in theinternodes after maceration treatments. The cell lengthincreased in the knat7 mutant but decreased in theKNAT7-OE lines when compared to the correspondingwild-type plants (Fig. 8A), implying that in addition tothe KNAT7-NAC31module, the KNAT7-GRF4modulealso functions in sclerenchyma fiber cells of internodes.

Figure 6. Validation of the KNAT7modulesin developing internodes and panicles. A,Transcription levels of KNAT7, NAC31,andGRF4 in different organs, including thedeveloping internodes and spikelets fromthe developing panicles of Nipponbare,showing the expression levels relative torice HNR. Data represent the mean 6 SD

of three biological replicates. R, root; Sh,leaf sheath; L, leaf. B, Fresh hand-cut crosssections of the developing internodes. S1–S3,three young segments from the bottom up.The red arrows indicate the thickeningcell wall. Bar 5 50 mm. C, SEM graphs ofglumes in 5-, 10-, and 15-cm panicles.Bar 5 20 mm. D, Quantification of thelength of glume cells examined in C, Dataindicate the mean 6 SD (n 5 50 cells fromfive spikelets).

Figure 7. Expression profile of genes involved in wall strengthening and cell expansion. A, The transcription level of secondarywall TFs and biosynthesis genes in the developing Nipponbare internode segments. Rice TP1 was used for normalization of theexpression of CESA genes, and HNR was used for normalization of the expression of MYBs. The transcription levels at segment1 (S1) were considered as 1. Data represent the mean6 SD of three biological replicates. B, Transcription level of expansin genesin spikelets from developing Nipponbare panicles. Rice HNR was used for normalization. The transcription levels at 0.5 cmwere considered as 1. Data represent the mean 6 SD of three biological replicates.

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To test the phenotypes in panicles, we performedthese examinations in the fiber cells of grain glumes.The fiber cell length in grain glumes increased in theknat7 mutant but decreased in the KNAT7-OE lineswhen compared to the correspondingwild-type plants

(Fig. 8B). Changes in wall thickness of the fiber cells ingrain glumes were similar to changes in cell length(Fig. 8, C–E). Alterations in KNAT7 led to increasedcellulose content in the knat7 mutant but reduced cel-lulose levels in the KNAT7-OE lines when compared

Figure 8. KNAT7 modules control the growth of sclerenchyma fiber cells. A, Measurement of the fiber cell length in the inter-nodes of the indicated plants. Data indicate the mean 6 SD (n 5 200 cells from three individual plants). *P , 0.01 by Welch’sunpaired t test represents a significant difference from the corresponding wild-type plants. B, Measurement of the fiber cell lengthin the glumes of the indicated plants. Data indicate the mean 6 SD (n 5 120 cells from three individual plants). *P , 0.01 byWelch’s unpaired t test represents a significant difference from the corresponding wild-type plants. C and D, SEM graphs of fibercells from the glumes of the indicated plants. Bars 5 2 mm. E, Measurement of the sclerenchyma fiber cell wall thickness in theglumes of the indicated plants. Data represent means6 SD (n5 125 cells from three individual internodes of the indicated plants).*P , 0.01 by Welch’s unpaired t test represents a significant difference from the corresponding wild-type plants. F, Cellulosecontent in the glumes of the indicated plants. Data represent the mean6 SD (n 5 3 biological replicates). *P , 0.01 by Welch’sunpaired t test represents a significant difference from the corresponding wild-type plants. G and I, A cross section of younginternodes (G) and glumes (I). The colored dashed lines indicate the cells harvested by laser microdissection. Bars5 100 mm. FC,fiber cells; PC, parenchyma cells. H and J, RT-qPCR analysis of cells harvested in G and I to show the expression levels relative torice HNR. The transcription levels detected in PC were considered as 1. Data represent the mean 6 SD of three replicates.K Working model of rice KNAT7. KNAT7-GRF4 modulates fiber cell expansion; the interaction represses the transcription ofexpansin genes activated byGRF4. KNAT7-NAC31 controlswall thickening; the interaction represses the expression of secondarywall regulatory TFs and biosynthetic genes. Therefore, rice KNAT7 integrates regulatory pathways of cell expansion and wallstrengthening to coordinate fiber cell growth. ZH11, Zhonghua11; NP, Nipponbare; L, line.

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to the corresponding wild-type plants (Fig. 8F), sug-gesting that KNAT7 modules regulate fiber cell growthin the panicles.

To obtain molecular support for the above conclu-sions, we investigatedwhetherKNAT7 and its partners,NAC31 and GRF4, are expressed together in fiber cells.Sclerenchyma fiber cells and parenchyma cells wereharvested from the young internodes and glumes bylaser microdissection (Fig. 8, G and I). RT-qPCR anal-yses performed in these cells revealed that the threegenes are consistently predominantly expressed in fibercells of the internodes (Fig. 8H), which was similarto the expression profiles of KNAT7 and NAC31 inglumes (Fig. 8J). Although GRF4 was equally tran-scribed in both cell types in glumes (Fig. 8J), the threegenes were coexpressed in fiber cells in the two ex-amined organs. These results suggest that KNAT7 isan integrative regulator in the control of cell expansionand wall thickening in fiber cells.

DISCUSSION

KNAT7 is a widely studied Class II KNOX protein.Although studies in several plant species have revealedthat KNAT7 governs secondary wall thickening, itsfunction differs among species. Arabidopsis KNAT7can repress lignin biosynthesis in the interfascicularfiber cells (Brown et al., 2005; Zhong et al., 2008; Liet al., 2012) and activate xylan production in xylarytissues (He et al., 2018). The mechanism underlyingKNAT7 function remains controversial. Rice has KNOXClass II members similar to those of Arabidopsis, andrice KNAT7, the only rice ortholog of ArabidopsisKNAT7, has an unknown function. In this study, wefound that rice KNAT7 interacts with distinct TFs tocoordinate cell expansion and wall stiffness.

Rice KNAT7 harbors all of the conserved domainsof KNOX proteins, similar to the Arabidopsis ortho-log (Ehlting et al., 2005; Persson et al., 2005), and it iscoexpressed with secondary wall biosynthetic en-zymes and TFs, indicating its role in wall thickening.That putative role in wall thickening has been cor-roborated in this study, as the rice knat7 mutant hadthickened secondary walls and the OE plants hadthinner walls. Cell wall composition analyses providedfurther support. Although rice KNAT7 possesses gen-eral TF features, it failed to induce the transcriptionof downstream TFs and secondary wall biosyntheticgenes, based on the results of transactivation activityanalyses. Interestingly, we found that rice KNAT7interacts with NAC31, an upstream master regulator ofsecondary wall thickening (Huang et al., 2015), therebyrepressing the expression of the NAC31 downstreamgenes MYB61 and MYB103. This finding places riceKNAT7 in the upstream hierarchy of the secondarywall regulatory network, which is distinct from pre-vious reports. Studies in several plant species havefound that KNAT7 can regulate secondary wall forma-tion in various ways (Brown et al., 2005; Li et al., 2012;

Gong et al., 2014; He et al., 2018), depending upon itstarget genes and interacting partners (Li et al., 2012;Zhong andYe, 2012). The divergent regulatory networkfor secondary wall biosynthesis among different plantspecies might be one of the reasons why KNAT7 func-tion varies in different plant species and in specific celltypes. Rice KNAT7 is hence an upstream regulator insecondary wall thickening. However, the possibilitiesthat this protein can transcriptionally regulate cell wallTFs or be regulated by other hierarchical TFs are notexcluded in rice.

More interestingly, we found that the rice knat7mutant and KNAT7-OE plants differed from eachother in grain size, which resulted from altered cellsize. Gene expression analyses revealed that severalexpansin genes proposed to facilitate cell expansionshowed different transcript levels among the KNAT7-modulated plants. Rice KNAT7 was further found tointeract with GRF4, a major quantitative trait locuscontrolling grain size by regulating cell expansion(Che et al., 2015; Duan et al., 2015; Li et al., 2018). Thisinteraction suppresses the expression of the expansingenes that are activated by GRF4. Hence, KNAT7 hasan unexpected role in controlling cell size. Althoughprevious studies have proposed that the roles of KNAT7might vary in different tissues and cell types (Li et al.,2012; He et al., 2018), its role in coordinating cell ex-pansion and wall thickening has not yet been found inother plant species.

Wall stiffening generally proceeds along with cellexpansion (Cosgrove, 2005; Huang et al., 2015). Cor-rect processing of the two cellular events is largelydependent upon formation of combinatorial modules,such as KNAT7-GRF4 and KNAT7-NAC31, at the righttime and the right place. Investigating when and wherethese modules form not only provided the in plantasupport for KNAT7 functions, but also offered a betterunderstanding of cell morphogenesis. Examining theexpression of KNAT7 and its interacting partnersNAC31and GRF4 in the developing internodes and pani-cles revealed the spatiotemporal regulation of theKNAT7-GRF4 and KNAT7-NAC31 modules. Basedon the expression patterns of the downstream genesduring internode and panicle development, the KNAT7modules were found to repress cell expansion and wallthickening. Wall-thickness and cell-size alterations infiber cells from internodes and glumes suggest thatboth regulatory modules can function in one cell type,which was further corroborated by coexpression anal-yses in the distinct cell types. Therefore, rice KNAT7can integrate regulatory pathways in fiber cells. KNAT7interacts with GRF4 to modulate cell expansion bysuppressing the transcription of expansin genes, whileit binds NAC31 to control wall strength by repres-sing wall-thickening regulatory pathways (Fig. 8K).This study suggests that KNAT7 plays a role in cellmorphogenesis, although further studies on how thesetwomodules cooperatively act in fiber cells are required.As the roles of KNAT7 are diverse, more KNAT7-interacting partners are expected to be found in plants.

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Hence, spatiotemporal characterization of KNAT7 andits cofactors is of great significance for understand-ing how KNAT7 functions and how cell growth isprogrammed.Coordination of cell expansion and wall strengthen-

ing occurs in response to internal and environmentalsignals. Our data demonstrate that rice KNAT7 playsrepressive roles in cell expansion and wall thickeningby forming distinct protein complexes. Thus, KNAT7 isa combinatorial regulator of plant cell growth. Serv-ing as an integrative regulator, KNAT7 can respond tovarious internal and external cues. The presence ofvarious regulatory elements in the promoter region(Supplemental Table S3) solidified the hypothesis thatKNAT7 has the potential to integrate multiple devel-opmental and environmental demands to coordinatecell size and wall stiffness. Our study offers a mech-anistic view for combinatorial modulation of plantcell growth and may provide a tool for the synergisticimprovement of lodging resistance and grain yieldin crops.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All rice (Oryza sativa) plants used in this study, including the wild-typeplants, knat7, and the KNAT7-OE plants, were grown in the experimentalfields at the Institute of Genetics and Developmental Biology in Beijingand in Lingshui, Hainan Province, China, in different growing seasons.Etiolated rice seedlings for generating protoplast cells were grown in adark growth chamber at 28°C. Nicotiana benthamiana and Arabidopsis(Arabidopsis thaliana) plants used in this study were grown in a greenhouseunder a 16 h light/8 h dark photoperiod at 23°C.

To investigate the agronomic traits, about 24 of the knat7 andKNAT7-OEplants of the T1 generation were planted in the fields. While the plantsmatured, the representative plants were photographed and subjected to phe-notype investigation. Specifically, the grain length and width of 15 grains har-vested from five plants were measured by an electronic digital display Verniercaliper. The 200 fully filled seeds from five plants were used tomeasure the 1,000-grain weight. The plant height and panicle length were obtained by measuringthe major tillers from 15 plants.

Generation of the Rice Transgenic Plants

To generate the knat7 mutant, single-guide RNA (sgRNA) sequencestargeting KNAT7 (789‒807 bp) were designed using theCRISPRdirect database(http://crispr.dbcls.jp/). The targeting oligonucleotides were synthesizedand annealed to form sgRNA duplexes and inserted into the guide RNAvector pYLgRNA-OsU3 (Ma et al., 2016). PCR was performed to obtain theexpression cassettes of OsU3-sgRNA, which was then inserted into the plantbinary vector pYLCRISPR/Cas9-MH to produce the CRISPR/Cas9 construct(Ma et al., 2016). For generation of the KNAT7-OE plants, the full-length codingsequence of KNAT7 was cloned and inserted into the pCAMBIA1300 vectorbetween the rice Ubiquitin promoter and the Nopaline synthase (NOS) termina-tor. The resulting constructs were transfected into Agrobacterium tumefaciensstrain EHA105 and introduced into the wild-type varieties Zhonghua11 andNipponbare, respectively. The primers used for preparation of the constructsare summarized in Supplemental Table S4.

Bioinformatics Analyses

Coexpression analysis was performed using the RiceFREND database(http://ricefrend.dna.affrc.go.jp/). Rice CESA4 was chosen as a guide gene,and the Pearson correlation threshold valuewas set above 0.6. The phylogenetictree of KNAT7 homologs in rice and Arabidopsis was built using MEGA6

software (Tamura et al., 2013) with the neighbor-joining methods and 1,000bootstrap replicates. Alignment of the sequences of KNAT7 and its homologs inrice and Arabidopsis was conducted using Clustal X (Larkin et al., 2007). Thesequences were obtained from rice and Arabidopsis genome databases (http://rice.plantbiology.msu.edu) and the National Center for Biotechnology Infor-mation (http://www.ncbi.nlm.nih.gov/). The cis-acting regulatory elements inthe KNAT7 promoter region were analyzed using the PLACE database (http://www.dna.affrc.go.jp/PLACE/).

Microscopy

Fresh hand-cut cross sections of the developing second internodes fromNipponbare were prepared as previously described (Huang et al., 2015). Theautofluorescent signals of cell walls were viewed and photographed with 488nm excitation using a fluorescence microscope (Imager D2, Zeiss). For scanningelectronmicroscopy analysis, themature second internodes and grains from thewild-type, knat7, and KNAT7-OE plants were harvested and fixed in 4% (w/v)paraformaldehyde (Sigma-Aldrich). The internode and grain samples weresliced with Gillette razor blades. After dehydration through a gradient of eth-anol and critical point drying, the samples were sprayedwith gold particles andobserved with a SEM (S-3000N; Hitachi). To examine the glume cell size of thedeveloping spikelets andmature grains, the glume outer surfaces of 10 spikeletsand grains were sprayed with gold particles and observed with a SEM(S-3000N, Hitachi). To measure the cell length of sclerenchyma fibers, themature internodes and grain glumes were macerated with glacial acetic acidand hydrogen peroxide (v/v, 1:1) at 80°C for 12 h. The tissues were squashedand stained with cellulose binding dye Pontamine Fast Scarlet 4B (Sigma) andphotographed with 543 nm excitation using a fluorescence microscope (ImagerD2, Zeiss). The data were analyzed and displayed using the ImageJ software.

Cell Wall Composition Analyses

The second internodes and grain glumes from the ;10 mature wild-type,knat7, and KNAT7-OE plants were collected to prepare cell wall residues.The cell wall residues were treated with pullulanase M1 (Megazyme) anda-amylase (Sigma) in 0.1 M sodium acetate buffer (pH 5.0) for 20 h to removestarch. The destarched alcohol insoluble residues were hydrolyzed by 2 M

trifluoroacetic acid. The supernatants were collected and analyzed by gaschromatography mass spectrometry (Agilent) to determine the monosac-charide content as described (Zhang et al., 2009). The remains were furtherhydrolyzed in Updegraff reagent (acetic acid:nitric acid:water, 8:1:2, v/v).The cooled pellets were thoroughly washed and hydrolyzed with 72% (v/v)sulfuric acid. The cellulose content was examined using the anthrone assay.The lignin content was measured using the acetyl bromide method (Huanget al., 2015).

Subcellular Localization

The full-length coding sequence of KNAT7 was cloned and in-frame fusedwith GFP and inserted into the binary vector pCAMBIA1300 between theCauliflower mosaic virus (CaMV) 35S promoter and NOS terminator. Theresulting constructs were transfected into A. tumefaciens strain EHA105and infiltrated into the leaves of 4-week-old N. benthamiana plants. The GFPfluorescent signals were recorded with a confocal laser-scanning microscope(TCS SP5; Leica), and 2 mg/mL of 49,6-diamidino-2-phenylindole (DAPI,Sigma) was used to visualize the nuclei.

Transactivation Activity Assays

For the TF feature analysis, the full-length coding sequence of KNAT7 wasamplified and in-frame fused with the GAL4BD domain and cloned into thevector p2GW7-GAL4BD. VP16 was cloned into p2GW7-GAL4BD as a positivecontrol. The GUS gene was cloned into p2GW7 as a negative control. Theeffector constructs for function validation were prepared by amplifying theKNAT7, GRF4, and NAC31 genes using the primers shown in SupplementalTable S4 and cloned into the vector p2GW7.

The reporter ProGAL4:Luciferase was prepared by placing five repeats ofthe Saccharomyces cerevisiae GAL4 binding elements plus the minimal TATAbox region of the CaMV 35S promoter upstream of the firefly (Photinuspyralis) luciferase reporter gene. Four copies of the KNOX binding elementwere synthetized and inserted before the 53GAL4 motif to generate the

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reporter construct ProKNOX-ProGAL4:Luciferase. The reporter constructsfor function validation were obtained by inserting the promoters (2 kb upstreamof the ATG) of EXPB3, EXPB17, EXPA6, MYB61, MYB103, and NAC31 genes(Supplemental Table S4) before the luciferase in the pUC19 vector.

The protoplast cells prepared from 4-week-old Arabidopsis rosette leaveswere pairwise cotransfected with the resulting reporter and effector constructs.After a 16- to 20-h incubation, the transfected protoplasts were lysed andluciferase activities were recorded using a dual-luciferase reporter assaysystem (Promega). The Renilla reniformis luciferase gene driven by the CaMV35S promoter was included in each assay to monitor transfection efficiency.These experiments were performed three times.

Protein Interaction Analyses

For split-luciferase complementation assays, the coding sequences ofKNAT7,GRF4,NAC31, andGibberellin Insensitive Dwarf1 (GID1) were amplifiedand in-frame fused with the amino- or carboxyl-terminus of luciferase. Theresulting constructs were transfected into A. tumefaciens strain EHA105 andpairwise coinfiltrated into the leaves of 4-week-old N. benthamiana plants. In-teractions were visualized by the bioluminescence signal intensity capturedusing IndiGO software (Berthold). GID1 fused with the N and C termini of theluciferase protein was used to generate the negative controls.

Yeast two-hybrid assays were performed according to the manufacturer’sinstructions (Clontech). The coding sequences of KNAT7, GRF4, and NAC31were cloned and inserted into the pGBKT7 and pGADT7 vectors to gen-erate the bait and prey constructs. The resulting constructs of combina-tory tests were cotransformed into the yeast strain Y2HGold. The yeastcells were cultured on synthetic dropout (SD) medium that lacks Trp, Leu,His, and Ade at 30°C for 3–4 d. The interactions were determined based onthe yeast growth status.

BiFC analysis was conducted as described (Zhang et al., 2018). In brief, thecoding sequences of KNAT7, GRF4, and NAC31 were amplified and insertedinto pSPY vectors that contain either amino- or carboxyl-terminal yellow flu-orescence protein (YFP) fragments. A. tumefaciens strain EHA105 bacteriacontaining the constructs were pairwise infiltrated into the leaves of 4-week-oldN. benthamiana plants. The interactions were visualized by the fluorescent sig-nal intensity recorded by a confocal laser scanningmicroscope (Axio imager Z2;Zeiss). The nuclei were stained with 2 mg/mL DAPI. Coinfiltrations of theconstructs with the corresponding empty construct were used as negativecontrols.

For FRET analysis, KNAT7,GRF4,NAC31, andGID1 genes were cloned andinserted into the FRET vector. The resulting constructs were transfected intorice protoplasts that were prepared from 2-week-old etiolated rice seedlings.Acceptor photobleaching FRET experiments were performed as described(Xie et al., 2018). The 405-nm and 514-nm argon ion lasers were used toexciteCFP and YFP fluorescence, respectively. The acceptor YFP fluores-cence in the region of interest in the nucleus was bleached by using 50 times514 nm argon laser line at 100% intensity. After photobleaching, the FRETefficiency was calculated using the following formula: FRETeff 5 (I4 – I3) 3100/I4. I4 represents the CFP intensity after the photobleaching of YFP, andI3 indicates the CFP intensity before photobleaching. The background FRETefficiency was calculated by measuring the fluctuation of CFP fluorescencebefore photobleaching. Fluorescence was recordedwith a confocal laser-scanningmicroscope (Axio imager Z2; Zeiss).

For co-IP analysis, the coding sequences of KNAT7 and NAC31 were fusedwith GFP or FLAG tag and inserted into the pCAMBIA1300 vector betweenthe Ubiquitin promoter and the NOS terminator. The resulting constructswere introduced into the wild-type variety Nipponbare to generate GFP-KNAT7-OE and FLAG-NAC31-OE transgenic plants. The Ubi:FLAG-GRF4 orUbi:GFP-KNAT7 constructs were transiently transfected into rice protoplastsprepared from seedlings of GFP-KNAT7-OE or FLAG-NAC31-OE plants,respectively. After cultivating overnight, the total proteins were extractedusing protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM

EDTA, 0.1% [v/v] Triton X-100, 0.1% [v/v] NP-40, and 13 protease inhibitorcocktail) and incubated with 30 mL of anti-GFP agarose beads (MBL) at 4°Cfor 2 h. GID1 was used as a negative control. The immunoprecipitationswere eluted with 23 SDS loading buffer at 95°C for 5 min. Proteins wereseparated by SDS–PAGE and detected by immunoblotting analysis usinganti-FLAG and anti-GFP primary antibodies.

All the experiments for protein-protein interaction examinations were per-formed three times. The representative images are shown. The primers used forthe construct preparations are included in Supplemental Table S4.

Gene Expression

Different organs, including roots, leaf sheaths, and leaves of 2-week oldseedlings and 9-cm developing internodes and young panicles ranging from0.5 to 15 cm long were collected from Nipponbare plants. The 9-cm developinginternodes were cut into nine segments. All segments except for the ninthsegments, as well as the spikelets from the developing panicles, were subjectedto RNA isolation. Total RNA was extracted using Plant RNA Reagent(Invitrogen). Meanwhile, the 9-cm developing internodes and 10-cm youngpanicles were harvested from the KNAT7-modulated plants and subjected toRNA isolation. One microgram of total RNA was reverse transcribed toproduce cDNA using the PrimeScript RT Reagent Kit (TAKARA) accordingto the manufacturer’s instructions. RT-qPCR was performed on a cyclerapparatus (Bio-Rad CFX96) using the FastStart Universal SYBR GreenMaster (Roche). The data were analyzed by the 2-DCT method. The cellular-level expression pattern was performed according to the report (Zhanget al., 2018). In brief, the second and third internode segments andspikelets from 10-cm panicles were embedded in paraffin and subjected tolaser microdissection. The 15-mm-thick sections were prepared to collectthe epidermal sclerenchyma cells and parenchyma cells by the LMD 7000laser microdissection system (Leica). Total RNA was extracted using theRNeasy micro kit (QIAGEN) and applied for RT-qPCR analysis. RiceHeterogeneous nuclear ribonucleoprotein 27C (HNR) and Triosephosphateisomerise1 (TP1) were used as internal controls for normalization of theexpression of TFs and CESA genes, respectively. The primers for RT-qPCRanalysis were summarized in Supplemental Table S5. These assays wereperformed at least three times.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers Os01g54620 (CESA4); Os10g32980 (CESA7);Os03g21820 (EXPA6); Os03g60720 (EXPA7); Os10g40720 (EXPB3); Os04g44780(EXPB17); Os05g33730 (GID1); Os02g47280 (GRF4); Os03g03164 (KNAT7);Os01g18240 (MYB61); Os08g05520 (MYB103); and Os08g01330 (NAC31).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Sequence identity of rice KNAT7.

Supplemental Figure S2. Phenotypes of the KNAT7-modulated plants.

Supplemental Figure S3. Rice KNAT7 has transcription factor features.

Supplemental Figure S4. Transactivation activity analysis of rice KNAT7.

Supplemental Figure S5. KNAT7 interacts with NAC31 and GRF4.

Supplemental Figure S6. The mutated KNAT7 is unable to interact withNAC31 and GRF4.

Supplemental Figure S7. Anatomical analysis of the developing inter-nodes and glumes.

Supplemental Table S1. List of the TFs coexpressed with rice CESA4.

Supplemental Table S2. Compositional analysis of sugar content of wallresidues from the internodes of the KNAT7-modulated plants and thecorresponding wild-type plants.

Supplemental Table S3. Identifying the cis-acting regulatory elements inthe 2-kb promoter region of KNAT7.

Supplemental Table S4. Primers used for construct preparation inthis study.

Supplemental Table S5. Primers used for RT-qPCR analysis in this study.

ACKNOWLEDGMENTS

We thank Professor Yaoguang Liu (South China Agricultural University) forkindly providing the guide RNA expression cassettes and the binary CRISPR/Cas9 vectors, Professor Letian Chen (South China Agricultural University) forkindly providing the vectors for FRET analysis, and Lijun Zhang (Institute of

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Genetics and Developmental Biology, Chinese Academy of Sciences) for helpwith lignin analysis.

Received May 28, 2019; revised July 17, 2019; accepted July 17, 2019; publishedJuly 29, 2019.

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