<i>Rice GROWTH-REGULATING FACTOR7</i> Modulates Plant Architecture through Regulating GA and Indole-3-Acetic Acid Metabolism

Abstract

Plant architecture is a major factor that determines grain productivity in cereal crop species (Wang and Li, 2008). Rice (Oryza sativa) is a staple food for more than half of the global population, and rice plant architecture has been continually improved by breeders to achieve high productivity. Rice plant architecture is mainly defined by plant height, the spatial pattern of leaves, and tiller and inflorescence branching patterns (Khush, 1995). Plant height and tiller branching determine the biomass and harvest index, while the spatial pattern of leaves, including leaf shape and angle, influences the photosynthesis rate and therefore the accumulation of carbohydrates (Sinclair and Sheehy, 1999; Springer, 2010; Xing and Zhang, 2010). During plant architecture determination, several factors, especially plant hormones, have been reported to affect the development of the leaf angle, plant height, and tiller number, thus modulating rice plant architecture. Phytohormones regulate many physiological processes that largely influence growth, differentiation, and development (Luo et al., 2016). Auxins and cytokinins mainly control the size and number of plant leaves, culms, inflorescences, and grains by regulating cell size and number (Schaller et al., 2015; Lavy and Estelle, 2016). Auxins also negatively control lamina joint inclination via asymmetric adaxial-abaxial cell division (Zhang et al., 2015). Consistent with these findings, the auxin early response gain-of-function rice mutant leaf inclination1, which encodes the indole-3-acetic acid (IAA) amido synthetase OsGH3-1, showed reduced free auxin levels and enlarged leaf angles due to stimulated cell elongation on the adaxial side of the lamina joints (Zhao et al., 2013). Overexpression of AUXIN RESPONSE FACTOR19 (OsARF19) resulted in an enlarged leaf angle via an increased expression level of GH3s and decreased free IAA content (Zhang et al., 2015). GAs control plant height and tillering by regulating cell elongation and cell division (Kobayashi et al., 1988; Magome et al., 2013; Gao et al., 2016). Blocking the synthesis of GA reduces plant height and therefore increases lodging resistance (Sasaki et al., 2002; Chen et al., 2015). Thus, the integration of multiple phytohormone signaling pathways will appropriately coordinate plant architecture development. Increasing evidence indicates that microRNAs (miRNAs) also participate in hormone synthesis or signal transduction and plant development (Tang and Chu, 2017). miR396 is one of the most conserved miRNA families in monocots and dicots (Liu et al., 2009). Studies have shown that repressing the expression of OsmiR396 in miR396 mimicry (MIM396) transgenic lines resulted in enlarged panicles (Gao et al., 2015). GROWTH-REGULATING FACTORs (GRFs), the targets of OsmiR396, are conserved plant-specific transcription factors. In rice, the GRF family comprises 12 members. All GRF genes have the conserved QLQ and WRC domains in their N-terminal regions. The QLQ domain is essential for protein-protein interaction, and the WRC domain contains a functional nuclear localization signal (Choi et al., 2004). GRFs interact with small cofactors called GRF-INTERACTING FACTORs (GIFs) to form a functional complex to regulate plant growth and development (Kim and Kende, 2004). GIFs have been reported to participate in cell proliferation during leaf development, root meristem homeostasis, and grain size determination (Kim and Kende, 2004; Li et al., 2016; Ercoli et al., 2018). In rice, the GIF family is composed of three members: OsGIF1, OsGIF2, and OsGIF3. OsGRF6 interacts with these three OsGIFs to regulate inflorescence architecture (Liu et al., 2014; Gao et al., 2015), OsGRF10 interacts with OsGIF1 and OsGIF2 to regulate floral organogenesis (Liu et al., 2014), and OsGRF4 interacts with OsGIF1 to regulate grain size (Li et al., 2016). These findings highlight the crucial roles of the OsmiR396-OsGRFs-OsGIFs module in determining the complexity of the regulatory network of rice growth and development. In this study, we report that OsGRF7 and OsmiR396e form a molecular node that regulates plant architecture via hormone-related genes, including OsARF12 (Wang et al., 2014; Li et al., 2020) and the cytochrome P450 gene OsCYP714B1 (Magome et al., 2013), which are involved in the auxin signaling pathway and GA synthesis, respectively. In vivo and in vitro assays indicate that these genes are directly regulated by OsGRF7. OsGRF7 also alters the contents of corresponding endogenous phytohormones and sensitivity to exogenous phytohormones. Our study demonstrates that OsGRF7 is a critical regulator of the shaping of plant architecture through GA and IAA signaling networks. A previous study showed that OsmiR396 regulates rice inflorescence branching and grain yield by modulating the expression of OsGRF6 (Gao et al., 2015). Here, we observed that MIM396 transgenic lines presented compact plant architecture. The leaf angle of the MIM396 transgenic lines was reduced by 26.9% at the seedling stage compared with that of the wild-type YuetaiB (YB; Supplemental Fig. S1, A and B). A similar phenotype was also observed for the second and third fully expanded leaves from the top of MIM396 plants at the tillering stage (Supplemental Fig. S1, C–E). Corresponding to the significant decrease in the abundance of OsmiR396 family members in MIM396-3 transgenic lines (Supplemental Fig. S1F), the expression levels of GRF family members, especially OsGRF7, were significantly increased (Supplemental Fig. S1G). To elucidate the functions of OsGRF7, we performed a genetic transformation of OsGRF7 and found that seven out of 13 OsGRF7 overexpression (GRF7OE) lines and four out of six OsGRF7 RNA interference (RNAi; GRF7RNAi) lines showed altered plant architecture (Fig. 1, A and B). Compared with that of the wild type, the plant height of the GRF7OE lines was reduced by ∼20% (Fig. 1A; Supplemental Table S1), corresponding to the contraction of each internode in the GRF7OE lines (Supplemental Fig. S2, A and B). Phenotypic characterization. A, Gross morphology of wild-type (WT), GRF7OE-1, and GRF7RNAi-1 plants. Bars = 15 cm. B, Comparison of the flag leaf angles (top row) and the top second leaf angles (bottom row) OsGRF7 transgenic Bars = cm. (top row) and (bottom row) of in GRF7OE-1, and GRF7RNAi-1 plants. Bars = of the of OsGRF7 transgenic (top row) and (bottom row) of the lamina joint of flag in the top are enlarged in the indicate adaxial side cell in of the lamina Bars = side cell of the of lamina In and are of indicate significant by The flag leaf angles of the wild the GRF7OE-1, and transgenic lines were and (Fig. Supplemental Table the plant height, leaf angle, leaf and leaf were with the expression levels of OsGRF7 in the transgenic plants (Supplemental Fig. that OsGRF7 regulates plant architecture in a GRF7RNAi-1 with MIM396-3 and found that the leaf angles were significantly decreased in the compared with in GRF7RNAi-1 (Supplemental Fig. A and B). The fully expanded leaf angles from the top of the decreased from in the GRF7RNAi-1 transgenic lines to in the and the third fully expanded leaf angles decreased from in the GRF7RNAi-1 transgenic lines to in the (Supplemental Fig. and that is involved in leaf angle To the of OsGRF7 in rice plant architecture determination, OsGRF7 lines were the second of OsGRF7 (Supplemental Fig. we four lines in which OsGRF7 was (Supplemental Fig. Supplemental Table Consistent with the leaf angles of the transgenic the angles of the second leaves of and were and (Supplemental Fig. which are significantly than of the wild-type the plant height of we found that the plant height of lines was significantly increased compared with the wild (Supplemental Fig. and These that OsGRF7 is a critical node for plant architecture determination in GRFs are conserved and plant-specific transcription factors. of the GRFs from rice and showed that OsGRF7 was to the and (Supplemental Fig. the expression of in the OsGRF7 transgenic we found that OsGRF7 was multiple showed increased expression especially and (Supplemental Fig. the and similar expression patterns with OsGRF7 (Choi et al., that these GRFs for the of OsGRF7 in the and To the of OsGRF7 in the control of plant we observed the of the and lamina The showed that plants more cell in the internode than wild-type and GRF7RNAi-1 plants (Fig. that the internode of the plants were also than of the wild and GRF7RNAi-1 (Fig. 1, and and the of the plants was significantly increased compared with that of the wild (Fig. corresponding to the and of the (Fig. The leaf angle increases the lamina joint from the leaf the lamina joint growth from lamina joint to the of the flag we found that the transgenic lines showed enlarged during the lamina joint development while GRF7RNAi-1 and showed the phenotype (Supplemental Fig. of and of the lamina joints compared with the wild-type and GRF7RNAi-1 the plants showed adaxial cell proliferation and cell elongation (Fig. 1, and that overexpression of OsGRF7 the cell division on the adaxial side of the lamina joint and thus increased cell on the adaxial side that lamina joint (Fig. and the compact phenotype of plants. GRFs are to regulated by miR396 et al., 2015). Here, we found that OsmiR396 directly OsGRF7 in vivo at the the OsmiR396 (Fig. we performed expression assays of OsGRF7 the and the in rice the of (Fig. transcription showed that the level of OsGRF7 significantly decreased OsGRF7 was with which with the decrease in OsGRF7 the of with of OsmiR396 members (Supplemental Fig. These that OsGRF7 is by OsGRF7 is mainly by OsmiR396e and in A, The OsGRF7 to The corresponding to the of the OsGRF7 by of and the of corresponding to the is shown by the of 12 have an OsmiR396 The in are in The indicate OsmiR396 and OsGRF7. The indicates the QLQ the indicates the WRC and the indicates the of OsGRF7. B, of OsGRF7 with in transgenic rice and control with or the respectively. was a The were by of expression levels of OsGRF7 and were by in inflorescence flag meristem and lamina expression patterns in transgenic plants. are 1, small to inflorescence at and root flag lamina joint of the Bars = expression levels of OsGRF7 in with was an are of three In rice, are that expression and 2014), that each of have To the regulatory of OsmiR396 members on OsGRF7, we in transgenic rice assays that at the OsmiR396 level (Supplemental Fig. the OsGRF7 content decreased the OsmiR396e (Fig. OsmiR396e the expression pattern of OsGRF7 in (Fig. that OsmiR396e is mainly for the of OsGRF7. the expression pattern of OsGRF7, and the showed that OsGRF7 was in the lamina and in and (Fig. corresponding to the that OsGRF7 was at levels in (Fig. The expression pattern of OsGRF7 was with roles in plant height, leaf size and angle, and tiller expression of the in rice showed that OsGRF7 was in the (Supplemental Fig. which is with the nuclear localization pattern in transgenic lines (Supplemental Fig. that OsGRF7 functions in the is in with the of the OsGRF7 in the expression (Supplemental Fig. and the of the OsGRF7 with GIFs (Supplemental Fig. and and in (Kim and Kende, 2004). These that OsGRF7 and OsGIFs to gene expression in the To elucidate OsGRF7 influences plant we performed by the transgenic In the more of than of the leaves or the more of the lines were to the targets and of OsGRF7. the with a of and with a of with the we and and of the were in the of genes in and (Fig. that OsGRF7 functions mainly in the of gene with nuclear localization (Supplemental Fig. A and B). These were to the genes, therefore to and genes (Supplemental and and were found with genes that for in and in (Fig. of the OsGRF7 A, of in the rice B, with in or by and of OsGRF7 in The indicates the OsGRF7 of OsGRF7 and was the in the of OsCYP714B1 and indicate = of in the of OsCYP714B1 and The was the of of a and expression levels of OsCYP714B1 and OsARF12 in the lamina joint of OsGRF7 transgenic lines were with was an and OsGRF7 OsCYP714B1 and OsARF12 in In to are of three indicate significant by and and of OsGRF7 and the of OsCYP714B1 and and were The or of in the is indicate the of on the we performed a of the an = was (Fig. which was by an in vitro (Fig. and these genes with we plant genes (Supplemental Table These genes four plant hormone-related genes, transcription factors, and of were to plant hormones, especially OsARF12 and which showed by OsGRF7, by (Fig. the in vivo of OsGRF7 with from OsARF12 and OsCYP714B1 (Fig. the of OsARF12 and OsCYP714B1 were in transgenic lines (Fig. and OsGRF7 the transcription of OsARF12 and OsCYP714B1 by their in assays (Fig. and the of OsGRF7 to from OsARF12 and OsCYP714B1 (Fig. and These that these genes are the targets of OsGRF7. the of the genes by and found that the of genes and and genes and several of the wild type, GRF7OE-1, and that OsGRF7 the expression of these genes (Fig. also the of OsGRF7 on these genes through the in vivo (Fig. OsGRF7 the expression of the gene by these (Fig. and these the that OsGRF7 directly regulates auxin and GA signaling OsGRF7 regulates the expression of multiple A, expression levels of auxin and GA genes in the lamina joint of the OsGRF7 transgenic was an to the to the of of with from the of auxin genes and and GA genes and The of was a and OsGRF7 and in In to and are of three indicate significant by and To the functions of genes in plant architecture determination, we with in OsGRF7 genes, and the which has a in the of OsCYP714B1 (Supplemental Fig. presented increased expression of OsCYP714B1 (Supplemental Fig. A previous report that OsCYP714B1 in GA synthesis and that high expression of OsCYP714B1 the plant height by cell elongation (Magome et al., 2013). Here, we the plant height of and found that was reduced by to that of the wild (Supplemental Fig. and with the reduced plant height of the GRF7OE lines (Fig. the a in third transcription (Supplemental Fig. and an enlarged leaf angle, with the second leaf angle from the top of the by compared with that of the wild (Supplemental Fig. and which was similar to that which for the These showed that the phenotype of each mutant was a of the plant architecture of the OsGRF7 transgenic that these genes of OsGRF7 to control plant architecture in also the contents of GA and IAA in of GRF7OE-1, and wild-type plants. The showed that the content was increased in (Fig. we content of at this stage mainly the was at the while the level of was high at the stage (Kobayashi et al., In rice, and are from and is more than their contents an and an in to a the and the plant height (Magome et al., 2013). phytohormone contents and exogenous phytohormone sensitivity of OsGRF7 transgenic A and B, of GA and IAA in OsGRF7 transgenic with are of three indicate significant by the wild of IAA at the lamina joint of OsGRF7 transgenic and were Bars = of OsGRF7 transgenic by of phytohormones. The control was for IAA and Bars = cm. of of IAA on root in the wild type, GRF7OE-1, and are = three and plants of of on the second in GRF7OE-1, and GRF7RNAi-1 are = three and plants In and significant from multiple to we found that the IAA and contents of significantly increased (Fig. which is with the leaf angle of plants (Zhang et al., 2015). that the adaxial side of the transgenic lines more auxin than the GRF7RNAi-1 and lines (Fig. is critical in regulating the cell growth of leaves, and the increased auxin content on the adaxial side of the lamina joints in the transgenic cell which is with the increased cell on the adaxial side of the transgenic (Fig. 1, and These that OsGRF7 directly regulates the synthesis of endogenous GA and IAA in OsGRF7 transgenic lines and their to IAA and a with of we found that the root of OsGRF7 transgenic lines decreased significantly IAA while the root of the GRF7RNAi-1 and wild-type decreased more than that of the transgenic lines that the GRF7OE lines are to auxin than the wild (Fig. and GA to to the height of the wild-type and GRF7RNAi-1 plants with GA the second of was than that of the wild (Fig. and that GA the phenotype of the GRF7OE plants. is in with the that high expression of OsCYP714B1 will to an in a in the content of (Magome et al., OsCYP714B1 encodes which to in In these that GRF7OE lines are to exogenous phytohormone the targets of OsmiR396, are members of a plant-specific gene family and are involved in many plant leaf root development, and (Liu et al., et al., 2013). Increasing of have that GRFs regulate leaf size by cell proliferation and in plant species and rice et al., 2010; et al., 2015). In GRFs mainly participate in leaf and growth, and cell division by is for cell the adaxial-abaxial during leaf (Wang et al., Overexpression of and resulted in and leaves that were and than of the wild (Kim et al., In a mutant of to leaves and (Kim et al., and a mutant of to leaves than (Kim and that mainly in plant height and leaf development. and OsGRF10 have been to involved in the of plant development, especially in grain and development (Luo et al., et al., 2014; et al., 2015; Gao et al., 2015; et al., while their roles in plant architecture determination are The module growth and rice and overexpression of including OsGRF7, and the roles of OsGRF7 have been in et al., Here, we found that OsGRF7, in with OsGIFs (Supplemental Fig. a of rice plant including plant height, leaf and and leaf angle (Fig. Supplemental Table is in with the of the acid that OsGRF7 was the of and (Supplemental Fig. 12 members of the gene family have been in rice (Choi et al., 2004). and OsGRF10 were found to regulate plant height and leaf size et al., 2015; et al., 2015; Li et al., et al., et al., found that overexpression of OsGRF7 resulted in decreased plant height (Supplemental Fig. and altered leaf that this gene cell division and cell elongation in the and lamina joint (Fig. 1, plant height increased in transgenic lines to of GRF7OE The and GRF7OE lines by in expression of GRF genes OsGRF7 is by increased levels of and (Supplemental Fig. we out the that the that OsGRF7 negatively regulates GRF in OsGRF7 was mainly the of leaves and (Fig. Supplemental Fig. which cell and cell proliferation of through especially on the adaxial side of the lamina to reduced leaf angle and in transgenic that OsGRF7 a in plant architecture These findings the functional of genes in Plant regulate many physiological processes that mainly influence growth, differentiation, and development (Luo et al., 2016). have been found to a of also many processes et al., and Chu, 2017). These through their targets to genes involved in plant hormone and or synthesis (Tang and Chu, 2017). Here, we found that OsGRF7 directly regulate a of genes and and the hormone of GAs and (Fig. regulate the development of leaves, and lamina joints and shape rice plant architecture (Fig. A reduced auxin level an leaf angle due to the of cell elongation and of the division of on the adaxial a of auxin on leaf inclination (Zhao et al., 2013; et al., 2017). a high auxin level is at the lamina joint of transgenic lines (Fig. and which is with the increased expression of OsGRF7 in the lamina joints (Fig. and with the showed that auxin is and in a high auxin on the adaxial side of the lamina cell division repressing cell elongation in transgenic lines (Fig. These findings of the of the module in hormone and plant architecture for the of rice plant architecture by OsGRF7. In GRF7OE OsGRF7 with OsGIFs to the expression of hormone-related genes through to the synthesis of is and the synthesis of IAA is the GRF7OE plants a and compact plant architecture. plant has been the for the of lodging resistance in rice a to lodging et al., 2010). Here, GRF7OE lines decreased plant height and from decreased (Supplemental Fig. and increased (Fig. 1, and The size of the was significantly and negatively with the lodging index, and the size of the lodging resistance in rice (Zhang et al., 2016). that the GRF7OE lines in the and leaves, and the in the and leaves than in the wild and the GRF7OE lines the leaves (Supplemental Fig. that the leaves and of the and lines and the the plant Consistent with this auxin was mainly at the lamina joint and and an auxin from the was observed (Fig. that auxin the leaf and development and level of auxin is for cell differentiation, and and and also development to regulate leaf angle (Zhang et al., 2015). These in the and lines will the leaves to and the plants to with and In the high auxin content in the transgenic lines increased the size in the lamina joints and in the (Fig. 1, and Supplemental Fig. in leaf angles and which in for rice lodging The leaf angle of rice is an that the of plant architecture. leaf angles leaf to more decreased for the which is for In plant architecture is by crop breeders and increases plant thus the accumulation of leaf for grain and grain yield (Wang et al., 2018). The of genes leaf angle and plant height is to rice plant architecture. Our of the OsGRF7 gene of the molecular of plant architecture and the of OsGRF7 a for plant architecture and lodging Rice (Oryza was the transgenic in this The and a were from the Rice of et al., MIM396-3 is the in previous study (Gao et al., 2015). GRF7OE-1, and transgenic lines are the in previous study et al., The of the OsGRF7 overexpression and transgenic lines was for the All the rice plants in this study were in to or to during to To the plants were in each and the plants of each were to the The was each and the was cm. The plant height, number, flag leaf and and leaf angle were at the stage than of grains were The plant height was from the node to the flag leaf of the and the number was with the more than fully The flag leaf of the tiller was to the flag leaf and and the leaf was and with The of OsGRF7 was from the inflorescence of and the overexpression by the To transgenic of was a the of of OsGRF7 was the plant expression of OsGRF7 was the to the gene These were the and in this study are in Supplemental The for OsGRF7 was reported et al., 2015). RNA in the second Supplemental Fig. in which the was by the rice and the was by the The expression were and the et al., 2015). The was the wild-type the transgenic lines were to and The of lines was for the The of lines are in Supplemental Table Plant were in and and in were with for were and to the cell and cell were and to the of the RNA from inflorescence was with to RNA and to the was performed with the The of was and of the an of and was transformation rice were at for RNA The OsmiR396 were of the with in the from rice of transgenic lines were with of or by for the OsGRF7 was by an RNA was to the levels of RNA was and the to the was performed by with to the The gene was the were performed to the and three were performed for each were with and at All were observed with an lines were for the OsGRF7 was the OsGRF7 was with and and of and the OsGRF7, OsGIF1, OsGIF2, and were with and and of et al., 2004). Rice were et al., with in the the and of the were with an of were a To the the or OsGRF7 with and were and The were the were on or The of OsGIF1, OsGIF2, and OsGRF7 with and were the and the or of OsGRF7 with and was The and were and on for at and were on The transgenic was for assays to the of et with of than or than was and with for and in The were and with the The was and in for were with the by the mainly to the the to was and with the were to the rice et al., 2009). was with a of for The the was with a size to the level of the To the of were by the and the were to The in was with their was performed in for each and the were to the to the The was the of a were performed for each from each of and with and and with and and and OsCYP714B1 with and were from and a the gene by the and the thus the to assays were performed rice and the was to the with the gene the were performed for each The was with and and expression To were at to an was to a of and were for at were and the was by the were and by were by and the of of of and of of of of of and were to a of (Liu et al., 2014), for at on and to in and at at for was by the to of IAA and GA were a To endogenous GA and 15 of the OsGRF7 transgenic lines were in a with with in a were and in to the of the was with with of at for 12 the was at for 15 The was and to a and in of The was for the was of was performed in To lamina joints were for in at and to at for were and was out by the in and at high for three with for the were in and for at were in a and and with the of to each were in a at were to a of with a and for 15 with for 15 and with The were on each and these were for to in a at with for 15 were with an with a and observed with a et al., 2010). hormone were on and phytohormones. of growth, the of the root and the second were of were and plants for each were for of OsGRF7 in rice and 12 rice GRF and GRF were in et al., The acid were the The was et al., on the with were performed The was for the of each transgenic with the wild The was performed with to the and from this found in the the OsGRF7, OsGIF1, OsGIF2, and in this study have been in the The are Supplemental Phenotypic and molecular of MIM396 transgenic Supplemental Comparison of internode the wild and OsGRF7 transgenic Supplemental OsGRF7 expression levels and of the OsGRF7 transgenic Supplemental OsmiR396 and OsGRF7. Supplemental and molecular of OsGRF7 Supplemental of GRFs in and Supplemental of members in the OsGRF7 transgenic Supplemental of lamina joint from to Supplemental of OsGRF7 by Supplemental The transcription levels of the OsmiR396 family in transgenic rice were with Supplemental of OsGRF7 and Supplemental and molecular of and Supplemental Table of OsGRF7 transgenic Supplemental Table of transgenic Supplemental Table of genes with OsGRF7 Supplemental with by in Supplemental with by in Supplemental in this and for their critical and during the of this for in the

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