Brassinosteroid Signaling Pathways Interplaying with Diverse Signaling Cues for Crop Enhancement

The signaling pathways of brassinosteroids (BRs), a unique plant steroid hormone, are critically involved in a diverse range of plant growth and developmental processes as well as many important agronomic traits. Recent advances in the understanding of BR biosynthetic and signaling pathways in model organisms and crops have increased the feasibility of modulating BR responses in crop plants to enhance adaptation to various vulnerable environmental changes. In particular, the identification and functional analysis of BR signaling components in rice (Oryza sativa) present the possibility of their utilization to improve many agricultural traits involved in crop yields. In this review, we summarize recent advances and progress in the understanding of the BR signaling pathway and its interactions with diverse internal and external signaling cues. We also discuss how these physiological modulations of BR and the abundant signaling crosstalk can be applied to enhance rice productivity through the manipulation of plant architecture and fine-tuning of stress responses. Finally, we discuss how the complex regulation of BR signaling pathways could favor application in the molecular design of plant growth and development, precise breeding strategies, and cultivation methods for rice crop improvement.


Introduction
Brassinosteroids (BR) regulate a diverse spectrum of processes in plant growth and development as well as internal modulation in response to environmental fluctuation. Since they are essential in plant adaptation and seed plant evolution, the biosynthesis and signaling pathways have been identified in the model plant Arabidopsis, providing a comprehensive understanding of how BR synthesis is controlled and how the signaling pathways are coordinated during the plant's life cycle [1][2][3][4][5][6]. Moreover, BR controls a number of important agronomic traits including plant height, grain size, tillering, leaf angle, and environmental adaptations, exploiting the potential of the BR response controlling genes as an interesting target by which to enhance crop performance [7,8]. In this review, we will focus on (1) the BR signaling pathway, (2) BR control of growth and development, (3) BR action on environmental adaptation in both Arabidopsis and rice as model systems for dicot and monocot plants, respectively, and (4) the potential precise modulation of BR responses in order to enhance crop breeding.

BR Signaling Pathway in Dicot and Monocot Model System
Brassinosteroids were originally isolated from rape (Brassica napus) pollen in 1979 [9]. After identification of BR as a plant-specific steroid hormone, extensive genetic and biochemical studies revealed its signal transduction mechanism in Arabidopsis. In detail, the initiation of BR signaling is tightly mediated by a receptor-like kinase, BRASSINOSTEROID INSENSITIVE 1 (BRI1), and co-receptor kinase, BRI1-ASSOCIATED KINASE 1 (BAK1), induces several BR responses, such as cell elongation and lamina inclination. Interestingly, BR negatively regulates DLT expression through OsBZR1 activation, but DLT induces OsBZR1 to maintain BR signaling homeostasis [20,24]. The loss-of-function dlt displays the typical BR-deficient phenotype, namely, semi-dwarfism, erect leaves, and reduced tiller number, but overexpression of DLT induces increased BR sensitivity and a physiological BR response. The DLT is also involved in the BR biosynthetic pathway in the context of feedback regulation in controlling the expression of key BR biosynthesis enzymes D2 and DWARF, suggesting that DLT is another positive transcriptional regulator of BR signaling in rice [7,25].
The CCCH-type zinc-finger protein, LIC, is another important transcriptional regulator in rice BR signaling. The silencing and ectopic expression of LIC resulted in elevated and compromised physiological BR responses in rice, respectively. In addition to genetic evidence, the biochemical study of OsGSK2 with LIC also supports its role in the BR signal transduction pathway. The LIC directly interacts with OsGSK2 and is phosphorylated and subsequently accumulated in the cytosol. As a consequence, BR deprivation leads to the loss of the transcriptional activity of LIC and results in enhanced BR sensitivity as a feedback mechanism. Importantly, OsBZR1 directly binds to the promoter of LIC and represses its expression in a BR-dependent manner, indicating the antagonistic relationship between LIC and OsBZR1 in rice BR signaling. Moreover, this relationship seems to be manifested in differential responses to low or high BR concentrations; a low physiological concentration of BR preferentially activates BR signaling via OsBZR1, but a high BR concentration mainly functions to repress the BR response in the LIC-dependent signaling pathway, which potentially aids in rice adaptation via precise BR responses in development and stress resistance [26,27].
The OsGRF4 is another key player controlling BR-responsive gene expression in rice. OsGRF4 was originally characterized as a positive regulator of grain size and filling in rice through grain-size-associated quantitative trait loci (QTL) analysis. Interestingly, OsGSK2 directly interacts with and represses OsGRF4 transcriptional activity under the control of BR perception and miR396 also targets the OsGRF family, indicating that OsGRF4 is specifically involved in grain-related BR responses and is an integrator of diverse hormonal crosstalk in rice [28][29][30][31].
The OFP has been recently identified as a transcription factor that interacts with the DLT protein. In the absence of BR, the OsGSK2 kinase attenuates OFP's transcriptional activity, whereas BR induces the accumulation of OFP protein in the nucleus and positively regulates the BR response in rice in a DLT-dependent manner [32]. Interestingly, ectopic expression of OFP stimulates gibberellin (GA) inactivation enzyme expression, suggesting that OFP serves as the locus of crosstalk in the context of the BR-induced GA inactivation process in rice [8].
A recent study identified a rice-specific interaction partner of OsBZR1. The APETALA2 DNA-binding transcription factor RLA/SMOS1 forms a complex with OsBZR1 for full activation of BR signal transduction. OsGSK2 also phosphorylates and inactivates, but auxin stimulates RLA/SMOS1 expression, indicating possible crosstalk between BR and auxin signal transduction [33,34].
The CYC-U2 protein, U-type cyclin, is also an important OsGSK2 kinase substrate promoting cell division in the mesocotyl of rice. In the presence of BR, inactivation of OsGSK2 leads to mesocotyl cell division [35]. The RELATED TO ABI3/VP1-LIKE 1 (RAVL1) is another BR signaling component that is required for full activation of BR signaling. The expression level of OsBRI1 is regulated by RAVL1, which directly controls the BR synthesis genes D2, D11, and BRASSINOSTEROID DEFICIENT 1 (BRD1) through binding to the Ebox on its target gene's promoter region [7]. The loss-of-function of RAVL1 is manifested by semi-dwarfism, delayed germination of seedlings, and a dark green phenotype, supporting the notion of its positive effect on rice BR responses [36].
In general, the identification of canonical BR signaling pathway components in rice has greatly improved the understanding of the BR's functional mechanism for enhancing crop performance. It is noteworthy that OsBZR1 directly/indirectly interacts with a number of TF complexes, such as DLT, LIC, OsGRF4OFP, RLA1/SMOS1, and CYC-U4;1, and these interaction partners are also largely controlled by OsGSKs in an OsBRI1-dependent manner. In the context of different BR levels in specific tissues and stages, differential assembly combination of OsBZR1-interacting transcriptional regulators will generate a diverse series of BR responses or specific BR responses in different tissues/stages during the rice life cycle. RAVL1 is manifested by semi-dwarfism, delayed germination of seedlings, and a dark green phenotype, supporting the notion of its positive effect on rice BR responses [36].
In general, the identification of canonical BR signaling pathway components in rice has greatly improved the understanding of the BR's functional mechanism for enhancing crop performance. It is noteworthy that OsBZR1 directly/indirectly interacts with a number of TF complexes, such as DLT, LIC, OsGRF4OFP, RLA1/SMOS1, and CYC-U4;1, and these interaction partners are also largely controlled by OsGSKs in an OsBRI1dependent manner. In the context of different BR levels in specific tissues and stages, differential assembly combination of OsBZR1-interacting transcriptional regulators will generate a diverse series of BR responses or specific BR responses in different tissues/stages during the rice life cycle.

BR Control of Plant Growth and Development in Dicot and Monocot
At the beginning of post-embryonic development, germination initiates communication between the environmental conditions and endogenous growth. This process is mainly regulated by a phytohormone, abscisic acid (ABA), that negatively regulates the seed germination in Arabidopsis ( Figure 2) [37]. In contrast, BR promotes germination by inhibiting the ABA response. These antagonistic regulatory mechanisms are mediated by BIN2. In the presence of ABA, BIN2 directly phosphorylates and activates the bZIP transcription factor ABSCISIC ACID INSENSITIVE 5 (ABI5) to regulate the expression of ABA-responsive genes. However, BR inactivates BIN2 and leads to the activation of TFs, BZR1/BES1. BR-activated BES1 physically interacts with TOPLESS-HISTONE DEACETYLASE 19 (TPL-HDA19), forming the inhibitory complex. BES1-TPL-HDL19 targets and represses ABI3 and ABI5 by mediating the deacetylation. In conclusion, BR acts as a positive regulator of seed germination [38,39]. BR was found to carry out a key function in the regulation of cambium maintenance and vascular cell differentiation [40]. In the BR signaling cascade, BZR1 and BES1 redundantly promote

BR Control of Plant Growth and Development in Dicot and Monocot
At the beginning of post-embryonic development, germination initiates communication between the environmental conditions and endogenous growth. This process is mainly regulated by a phytohormone, abscisic acid (ABA), that negatively regulates the seed germination in Arabidopsis (Figure 2) [37]. In contrast, BR promotes germination by inhibiting the ABA response. These antagonistic regulatory mechanisms are mediated by BIN2. In the presence of ABA, BIN2 directly phosphorylates and activates the bZIP transcription factor ABSCISIC ACID INSENSITIVE 5 (ABI5) to regulate the expression of ABA-responsive genes. However, BR inactivates BIN2 and leads to the activation of TFs, BZR1/BES1. BR-activated BES1 physically interacts with TOPLESS-HISTONE DEACETYLASE 19 (TPL-HDA19), forming the inhibitory complex. BES1-TPL-HDL19 targets and represses ABI3 and ABI5 by mediating the deacetylation. In conclusion, BR acts as a positive regulator of seed germination [38,39]. BR was found to carry out a key function in the regulation of cambium maintenance and vascular cell differentiation [40]. In the BR signaling cascade, BZR1 and BES1 redundantly promote xylem and phloem cell differentiation. proliferation and differentiation in close interaction with the BR signaling pathway [41][42][43][44]. The TDR-TDIF signaling directly suppresses BES1 and BZR1 through the activation of the BIN2 kinase in the cambial region, which inhibits and facilitates xylem formation and cambium proliferation, respectively [44].
BR is known to be a key positive hormonal regulator of seed and grain development in both Arabidopsis and rice. In Arabidopsis, the BR biosynthesis-deficient mutant, de-etiolated 2 (det2), shows reduced seed size and fertility [45]. Among a number of genes controlling the seed and embryo/endosperm development, SHORT HYPOCOTYL UNDER BLUE 1 (SHB1), HAIKU 1 (IKU1), HAIKU 2 (IKU2), and MINISEED 3 (MINI3) are known to be the main regulators of seed size and weight through their interactions with canonical BR signaling [46][47][48][49]. These proteins act downstream of BZR1 under the control of the BR-BRI1-BIN2 phosphorylation cascade. On the other hand, BR also inhibits the expression of APETALA 2 (AP2), the floral homeotic gene, and AUXIN RESPONSE FACTOR 2 (ARF2), the key negative regulators of seed size and weight [50,51].
In rice, extensive reverse genetic screens isolated from a number of mutants identified a significant number of BR-related genes in these mutant pools [28,52]. In addition, several key players in BR signaling derived from QTLs support the notion of BR as an essential determinant of grain size, shape, and filling in rice. Among them, the loss-of-function Osbri1 mutant and OsGSK2 overexpression in rice lead to decreased grain size and weight [53]. The seed-size-associated QTL, GL2, which encodes the OsGRF4, also regulates the seed size by inducing the BR response. Antagonistically, GSK2 directly interacts with and inhibits OsGRF4, which results in a reduction in seed development and growth in rice [28]. In addition, GRAIN WIDTH 5 (GW5), a membrane-localized calmodulin-binding protein, regulates the grain width and weight, acting through a promoter of BR signaling [54]. In detail, the GW5-overexpressing plants in the OsGSK2 overexpression line (Go) background show increased grain length and hypersensitivity to exogenous BL treatment, indicating GW5 as a novel BR signaling component involved in grain development. GW5 directly interacts with and suppresses OsGSK2 activity by compromising the auto-phosphorylation of OsGSK2, which is required for its activation. Subsequently, GW5-mediated inactivation of OsGSK2 induces the BR response through the accumulation of the active form of OsBZR1 and DLT in the nucleus. Besides these developmental processes, grain filling contributes to the yield of cereal crops. The organ-specific enhancement of BR synthesis at the stem, leaf, and root, but not at the embryo or endosperm, increases the grain weight and number through increased carbon partitioning to the sink tissue (grain). This process is controlled by the OsBZR1 target, MYB-domain transcription factor CARBON STARVED ANTHER (CSA) [55,56].
The erect leaf and decreased lamina joint angle of rice are key agronomic traits involved in crop enhancement. BR is responsible for increasing the lamina joint angle through the differential elongation of adaxial/abaxial cells in this region. In detail, BR inhibits the protein accumulation and expression level of CYC-U4;1, consequently suppressing cell proliferation in the abaxial side of the lamina joint region through inactivation of OsGSK2 and activation of OsBZR1, respectively [57].

BR Crosstalk with the Environmental Signal in Dicot and Monocot
In Arabidopsis, BR appears to play the role of central integrator between internal signal and external stress/signal cues. Dark conditions or high air temperatures cause hypocotyl elongation due to integration with endogenous hormonal cues, BR, GA, and auxin. In detail, de-det2, a BR biosynthesis-deficient mutant, displays a defect in the form of dark-induced hypocotyl elongation, whereas bzr1-1D, a mutant that is hypersensitive to BR, suppresses the dark-induced hypocotyl elongation of det2. At the molecular level, BR-activated BZR1 forms a heterodimer with PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and directly regulates the expression of PACLOBUTRAZOLE RESISTANCE (PRE) family genes which are positive regulators of cell elongation [58]. In conclusion, these results suggest the crucial role of BR signaling in cell elongation and crosstalk with the stress response, especially under high-temperature conditions. Under drought stress, ABA stabilizes and activates the transcription factor RESPONSIVE TO DESICCATION 26 (RD26) through BIN2-induced phosphorylation. RD26 regulates the drought-stress-responsive genes and inactivates BES1 to inhibit growth. In contrast, under normal conditions, BR prevents BIN2 activity and BRactivated BES1 downregulates the expression of RD26 [59,60]. Heat stress, which prevents optimal plant growth, accompanies protein denaturation, increased membrane fluidity, and the production of reactive oxygen species (ROS), which all cause photoinhibition and photooxidative stress. Photoinhibition-and photo-oxidative-stress-induced overproduction of ROS results in cellular and molecular damage. However, low levels of ROS rather play a role in the plant stress response by acting with phytohormones, such BR, ABA, salicylic acid (SA), and jasmonic acid (JA). Under heat stress, BR upregulates the expression of RESPIRATORY BURST OXIDASE HOMOLOGS (RBOH) at an early stage, which functions as NADPH oxidase and leads to apoplastic accumulation of H 2 O 2 [61]. BR-induced H 2 O 2 triggers the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) pathway and ABA biosynthesis. In addition, BR primarily mediates the initial stress resistance response, but the prolonged response is regulated by ABA, which is triggered by BR [62]. Meanwhile, in the context of BR signaling, the loss-of-function mutant, bes1, is more sensitive to heat stress compared with WT [63]. In rice, previous studies elucidated the effect of BR in the pathogenspecific function of BR in rice. A brassinolide (BL) treatment experiment showed the broad positive effect of BR for resistance to a broad range of pathogens [64]. To a significant extent, the BL-treated plant rescues the Magnaporthe grisea (M. grisea)-induced lesions on the leaf blade and Xanthomonas oryzae-mediated damage. Moreover, BR induces hypersensitivity to some specific viruses, including the rice black-streaked dwarf virus (RBSDV), which causes a broad range of severe symptoms in rice [65]. Based on transcriptional profiling after RBSDV infection in rice, expression levels of OsDWRF4, OsCPD, OsBRI1, and OsBAK1 are dramatically decreased, while JA signaling-related genes are quickly upregulated. Consistent with this expression pattern, JA-treated rice displays increased resistance to RBSDV and reduced transcript levels of BR-related genes. Interestingly, the transcript levels of BR signaling components show little fluctuation in coi1-13, a loss-of-function mutation of the JA signaling coreceptor, O. sativa CORONATINE INSENSITIVE 1 (OsCOI1), suggesting that the virus-specific effect of BR signaling is suppressed by JA through OsCOI under stress conditions in rice [65]. The BR also regulates the immune response in the root by antagonistic action with SA and GA against the fungal pathogen Pythium graminicola (P. graminicola) [66].
Salinity is one of the major external stresses that severely affects plant development, growth, and seed production. Salt stress mostly impedes the yield of crops by limiting their optimal growth, affecting traits such as source organ size, plant height, grain size, grain weight, and grain quality. Interestingly, among the key salt stress-related hormones in rice, namely, ABA, auxin, and BR, the growth-promoting hormone BR enhances the salt tolerance through the action of OsSERK2, which behaves as a BR signaling co-receptor with OsBRI1 [31]. Interestingly, both the somatic embryogenesis receptor-like kinase 2 (serk2) knockout mutant and SERK2-overexpressing rice show increased grain size, but the SERK2overexpressing plant displays resistance to salt stress, suggesting a complex interaction network between BR's promotion of growth and stress tolerance. Moreover, in an analysis of rice T-DNA insertion lines that are associated with abiotic stress, the OsGSK1 was isolated as a negative regulator of BR signaling and a key component conveying potential BR-mediated stress resistance [19].

BR Application for Increasing Crop Yield in Rice
Recent studies have reported effects on the positive correlation between BR and crop yield increase [67]. Consistently, the physiological effects of BR on the regulation of many important agronomic traits, including the number and weight of grains and planting density, have been widely evaluated, especially in rice. It is well known that enhancing the endogenous BR content by overexpressing its biosynthetic-related genes or exogenous BR application is able to increase grain number and weight [55]. Rice plants overexpressing BR signaling components and Arabidopsis C-22 hydroxylase in the embryos or endosperms displayed BR responses such as enhanced bending of the lamina joint and increased grain size [55,68]. Conversely, many BR biosynthetic-or signaling-related rice mutants showed a dwarf shoot architecture, harboring small seed size and bearing seeds reduced in both length and width. The successful breeding of high-yielding semi-dwarf wheat and rice varieties has contributed significantly to the green revolution of these crops. The semidwarf varieties resulted in higher crop yields by enhancing seed biomass production and tolerance to the lodging of rice stems [69]. Many studies reported the critical role of BR in the elongation of the second internode. A BR-insensitive Osbri1 mutant (d61) and BR biosynthetic d2 and d11 mutants displayed dwarf phenotypes [18,25,53,70]. As mentioned above, planting density is also one of the most important agronomic traits for crop yield. The rice varieties with erect leaves enable more dense planting and show higher photosynthetic activity. BR and auxin are well-known hormones that determine the leaf angle of rice [71]. BR-defective rice mutants generally display erect leaf angles with dwarf shoots. RAVL1-mediated regulation of BR homeostasis through the downstream action of BRI1, D2, D11, and BRD1 in rice is critically involved in enlarged leaf angle [25,36]. Upstream BR signaling pathways are integrated into OsBZR1 to directly or indirectly regulate LIC, IBH1, and a rice U-type cyclin, CYC-U4;1, to increase lamella jointing cell elongation [27,72]. Similarly, GA-related d1 mutant (mutated in OsRGA1) also showed a stunted second internode phenotype with erect leaves, suggesting that a specific synergistic interplay between BR and GA takes place in the regulation of rice architecture [73][74][75].
BR signaling outputs are also positively connected with the improvement of grain size and filling. Tissue-specific overexpression of a BR biosynthetic Arabidopsis C-22 hydroxylase gene in grain tissues increased grain weight and grain number in rice. Similarly, the ectopic expression of BR positive regulators O. sativa INCREASE LEAF INCLINATION 1 (OsILI1) and OsILI4 served to enhance both grain size and bending of the lamina joint [68]. Consistently, BR-defective mutants including d61, d11, d2, and brd1 bear small seeds, suggesting the positive role of BR signaling in rice grain filling. Although the BR-defective erect rice mutants showed greater biomass production with higher planting density, the final grain yield did not increase significantly due to the small grain size [53]. However, recent studies have reported alternative approaches to overcome the small-grain phenotype of BR-defective mutants through co-suppression in the shoot by reducing the expression of endogenous OsBRI1. This mutant showed erect leaves and normal seed size, and thus it could be planted at high density. The estimated grain yield of this transformant was almost 30% higher than that of the wild type. A recessive dwarf4-1 mutant reduces bioactive BR content in shoots, resulting in erect leaves but normal reproductive development. Under high-density planting conditions, the grain yield of the dwarf4-1 rice mutant resulted in a nearly 40% enhancement [76]. These findings demonstrate that the yield of rice crop can be increased by generating erect-leaf plants with no defects in grain formation by specifically reducing the BR signaling activity in shoots rather than targeting the reproductive organs. These results collectively indicate that specific BR-related components could offer the possibility to modulate BR responses in order to obtain desirable agronomic traits to improve crop yield and environmental adaptation in rice. Agronomy 2021, 11, x 8 of 12

Perspective
Currently, BR signaling components in monocots and dicots are undergoing characterization with extensive forward-/reverse genetic screening. Here, we highlighted the current understanding of the signaling network which controls BR signaling in dicot and monocot model systems. The functional characterization of novel BR signaling components in the monocot boosts the breeding program with desirable and customized phenotypes in modern agriculture, such as semi-dwarfism, stress resistance, high yield, and erect leaves. These traits are all strongly associated with BR in cereals and serve as promising targets for enhanced crop yield. Therefore, functional characterization of new components in BR signaling, as well as investigation of their signaling network with endogenous/exogenous signaling cues, are essential fields of research for future crop breeding programs.

Perspective
Currently, BR signaling components in monocots and dicots are undergoing characterization with extensive forward-/reverse genetic screening. Here, we highlighted the current understanding of the signaling network which controls BR signaling in dicot and monocot model systems. The functional characterization of novel BR signaling components in the monocot boosts the breeding program with desirable and customized phenotypes in modern agriculture, such as semi-dwarfism, stress resistance, high yield, and erect leaves. These traits are all strongly associated with BR in cereals and serve as promising targets for enhanced crop yield. Therefore, functional characterization of new components in BR signaling, as well as investigation of their signaling network with endogenous/exogenous signaling cues, are essential fields of research for future crop breeding programs.

Conflicts of Interest:
The authors declare no conflict of interest.