
==== Front
Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00290-6
10.1016/j.xplc.2024.100982
100982
Review Article
Brassinosteroid biosynthesis and signaling: Conserved and diversified functions of core genes across multiple plant species
Zebosi Brian 12
Vollbrecht Erik vollbrec@iastate.edu
12∗
Best Norman B. norman.best@usda.gov
3∗∗
1 Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA
2 Interdepartmental Genetics and Genomics Graduate Program, Iowa State University, Ames, IA 50011, USA
3 USDA-ARS, Plant Genetics Research Unit, Columbia, MO 65201, USA
∗ Corresponding author vollbrec@iastate.edu
∗∗ Corresponding author norman.best@usda.gov
29 5 2024
09 9 2024
29 5 2024
5 9 10098213 3 2024
13 5 2024
28 5 2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Brassinosteroids (BRs) are important regulators that control myriad aspects of plant growth and development, including biotic and abiotic stress responses, such that modulating BR homeostasis and signaling presents abundant opportunities for plant breeding and crop improvement. Enzymes and other proteins involved in the biosynthesis and signaling of BRs are well understood from molecular genetics and phenotypic analysis in Arabidopsis thaliana; however, knowledge of the molecular functions of these genes in other plant species, especially cereal crop plants, is minimal. In this manuscript, we comprehensively review functional studies of BR genes in Arabidopsis, maize, rice, Setaria, Brachypodium, and soybean to identify conserved and diversified functions across plant species and to highlight cases for which additional research is in order. We performed phylogenetic analysis of gene families involved in the biosynthesis and signaling of BRs and re-analyzed publicly available transcriptomic data. Gene trees coupled with expression data provide a valuable guide to supplement future research on BRs in these important crop species, enabling researchers to identify gene-editing targets for BR-related functional studies.

This review summarizes recent research on brassinosteroids, a class of plant growth hormones that participate in several developmental processes. Phylogenetic analyses coupled with published RNA-seq data are used to characterize the conserved and diversified functions of genes related to brassinosteroid biosynthesis, catabolism, and signaling across multiple species, providing guidance for future genetic studies.

Key words

brassinosteroids
maize
rice
Setaria
soybean
Brachypodium
Arabidopsis
Published: May 29, 2024
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pmcIntroduction

Brassinosteroids (BRs) are a class of plant-specific steroidal hormones. Their functions in the regulation of plant growth and development have been studied in many plant species. BRs, initially described as “brassins,” were first extracted from Brassica napus (rapeseed) pollen as a crude lipid extract that, when mixed with lanolin, resulted in strong internode elongation of a bean plant grown in the light (Mitchell et al., 1970). The first structure of a BR, specifically brassinolide (BL), was determined in 1979 and established the growth-promoting BRs as polyhydroxyl lactones that commonly have a 5 α-cholestane skeleton, like that of animal steroids (Grove et al., 1979). BRs are present in all plant tissues and organs and have been identified in green algae as well as bryophytes, moss, lycophytes, gymnosperms, and angiosperms (Yokota et al., 1987; Bajguz et al., 2020).

BR function has been studied extensively in Arabidopsis thaliana (Arabidopsis) owing to the development of many mutants in different biosynthetic enzymes and BR signaling and response components. Some work has been published on BRs in other crop and model species, including Zea mays (maize), Oryza sativa (rice), Setaria viridis (Setaria), Brachypodium distachyon (Brachypodium), and Glycine max (soybean); however, there are still many unknown gene functions across species because of the lack of mutant lines. Investigating the conservation and/or diversification of these different gene families in eudicots and monocots is necessary for a broad understanding of BR gene functions across species. To that end, identification of gene homologs facilitates the selection of reverse genetic targets to investigate biological questions. To accomplish this, we conducted a comprehensive phylogenetic analysis of encoded BR gene products across Arabidopsis, maize, rice, Brachypodium, soybean, and Setaria; re-analyzed publicly available transcriptomic experiments across multiple tissues in these species; and presented these data together for BR biosynthesis, signaling, homeostasis, and response components. We selected genes on the basis of research showing their involvement in brassinosteroid biosynthesis, signaling, or catabolism in at least one of the presented species. To assist the reader, we have included in the text and figures an abbreviation of the specific genus and species we are describing before the gene name (i.e., At [(Arabidopsis], Zm [maize], Os [rice], Sv [Setaria], Bd [Brachypodium], and Gm [soybean]).

Brassinosteroid-independent biosynthesis pathway

Complex networks of biosynthetic pathways for BR production have been uncovered in plants. We focus in this article on the primary pathways leading to sterols, tracing through cycloartenol to active BRs. We describe this segment as the BR-independent pathway, as exogenous BR treatment is insufficient to rescue the corresponding mutant phenotypes. Other reviews treat more comprehensively a broader range of possible pathways leading to BRs, e.g., the mevalonate and non-mevalonate pathway branches upstream of sterols and the alternative pathway branch through cycloartenol and downstream of sterols (Bajguz et al., 2020). There are over 250 different sterol compounds in plants, and the first dedicated compound in the biosynthesis of these phytosterols is squalene (Schaller, 2003, 2004; Vriet et al., 2013).

Cycloartenol synthesis involves the monooxygenation of squalene to squalene epoxide by SQUALENE EPOXIDASES (SQEs) and cyclization of squalene epoxide to either cycloartenol by CYCLOARTENOL SYNTHASE1 (CAS1) in plants (Supplemental Figures 1 and 2B) or lanosterol by LANOSTEROL SYNTHASE (LAS) in animals and fungi (Rasbery et al., 2007; Desmond and Gribaldo, 2009; Laranjeira et al., 2015). The synthesis of cycloartenol by CAS1 and lanosterol by CAS1 and LAS produces cholesterol, ergosterol, and phytosterol in mammals, fungi, and some plants, respectively (Suzuki et al., 2006; Ohyama et al., 2009).

Cycloartenol to 24-methylene-cycloartanol

In plants and fungi, phytosterols have extra alkyl groups at C-24, regulated by S-adenosylmethionine-dependent STEROL C-24 METHYLTRANSFERASEs (SMTs) (Diener et al., 2000; Holmberg et al., 2002). In contrast to fungi, which have a single methyl group at C-24, higher plants undergo sequential C-24 methylation reactions catalyzed by distinct SMT genes. SMT1s, conserved across several kingdoms, convert cycloartenol to 24-methylene-cycloartenol, a precursor of campesterol (CR) and sitosterol synthesis. SMT2 and SMT3, which diverged from the ancestral SMTs, catalyze the second methylation of 24-methylenelophenol to 24-ethylidenelophenol, required for sitosterol synthesis (Carland et al., 2010; Haubrich et al., 2015; Nakamoto et al., 2015).

Phylogenetic analysis showed expansion of both the SMT1 and SMT2/SMT3 gene families (Figure 1A). Across the clades, SMT1 genes are more highly expressed than SMT2/SMT3s in most tissues. In Arabidopsis, AtSMT1 is primarily expressed in developing embryos, shoots, and roots. AtSMT2 and AtSMT3 are moderately expressed in most tissues (Figure 1A; Diener et al., 2000). Like those of Arabidopsis, SMT1 genes in Brachypodium, rice, and maize are extensively expressed in most tissues, whereas SMT2s/SMT3s are expressed in the inflorescence.Figure 1 Phylogeny and transcript abundance across different tissues of SMT, SMO1, and SMO2 phytosterol biosynthetic enzyme families.

Maximum-approximate-likelihood phylogenetic trees of (A) SMT, (B) SMO1, and (C) SMO2 amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp) are shown. The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

The SMT1 and SMT2/SMT3 genes have been shown to regulate several developmental processes, such as embryo morphogenesis, cotyledon vein patterning, apical dominance, auxin response, and reproductive and root development (Carland et al., 2010; Nakamoto et al., 2015). The Atsmt1 mutants exhibit reduced plant stature with aberrant embryo morphogenesis and aborted embryos (Diener et al., 2000). Consistent with their expression data, AtSMT2 and AtSMT3 function collectively to regulate plant development. AtSMT2 and AtSMT3 single mutants exhibit a mild phenotype, and their double mutants display enhanced developmental defects such as extreme dwarfism, aberrant root development, deformed cell shape, and disorganized tubulin orientation with impaired gravitropism and aberrant localization of auxin efflux carriers (Carland et al., 2010; Nakamoto et al., 2015). Functional studies will be needed to understand the roles of these SMTs in other species.

24-Methylene-cycloartanol to cycloeucalenol and 24-methylenelophenol to 4α-carboxy-5α-cholesta-7,24-dien-3β-ol

Demethylation of (first) 24-methylenecycloartenol into cycloeucalenol and (second) of 24-methylenelophenol into episterol is catalyzed by two non-sequential independent STEROL C-4 METHYL OXIDASES (SMO1 and SMO2), respectively, in contrast to fungi and animals in which the two C-4 demethylation reactions are sequentially catalyzed by a single SMO (Sonawane et al., 2016; Song et al., 2019a). The SMO1 gene family has between two and five genes in each species examined. In Arabidopsis, AtSMO1-1 and AtSMO1-2 are widely and jointly expressed in most tissues (Figure 1B; [Song et al., 2019a]). The expression patterns of SMO1 genes in maize and Brachypodium suggest specialization across tissues such as the embryo, young inflorescence, and mature inflorescence. In rice, the two genes LOC_Os10g39810 and LOC_Os03g01820 show differential expression, with LOC_Os03g01820 more widely expressed. In soybean, SMO1s are ubiquitously and moderately expressed across tissues. In the SMO2 family (Figure 1C), Brachypodium has a single gene, and the other species have two SMO2s. SMO2 genes show broader tissue expression patterns than their SMO1 paralogs, and the degree of overlapping expression within species suggests a corresponding functional redundancy.

Functional characterization is restricted to Arabidopsis, in which SMO1 and SMO2 single mutants had no obvious developmental defects, whereas both Atsmo1-1; Atsmo1-2 and Atsmo2-1; Atsmo2-2 double mutants resembled auxin-defective mutants and produced nonviable embryos with aborted shoot apical meristems, suppressed cytokinin, and accumulated auxin levels owing to impaired auxin transport (Song et al., 2019a; Zhang et al., 2016a). The embryo lethality of the double-mutant was partially rescued by auxin or cytokinin treatment and AtYUCCA9 overexpression (Zhang et al., 2016a; Song et al., 2019a). Thus, SMO1s and SMO2s are vital for embryogenesis and regulation of auxin production and transport (Zhang et al., 2016a; Song et al., 2019a). Functional characterization in other species is required to examine the broader functional diversification or conservation of these genes.

Cycloeucalenol to 4α-methylergostatrienol

CYCLOPROPYLSTEROL ISOMERASE1 (CPI1) and OBTUSIFOLIOL 14α-DEMTHYLASE (CYP51G1) catalyze the two-step conversion of cycloeucalenol to obtusifoliol and obtusifoliol to 4α-methylergostatrienol, respectively (Men et al., 2008). CPI1 is restricted to land plants, as fungi have no CPI1 homolog (Lovato et al., 2000; Sonawane et al., 2016). CPI1 is a single-copy gene in Arabidopsis, Brachypodium, maize, Setaria, and rice and a two-copy gene in soybean (Figure 2A). CPI1 genes showed distinct tissue-specific expression patterns across species, suggesting corresponding species-specific sterol utilization. Functional studies indicated that CPI1 regulates several aspects of plant growth and development, such as embryo morphogenesis, root development, and auxin distribution, and their mutant resembled SMO1/SMO2 double mutants (Men et al., 2008; Wang et al., 2021). No functional studies of CPI1 genes have been reported in monocots or soybeans.Figure 2 Phylogeny and transcript abundance across different tissues of CPI, CYP51G1, HYD1, HYD2, and 3βHSD/D phytosterol biosynthetic enzyme families.

Maximum-approximate-likelihood phylogenetic trees of (A) CPI, (B) CYP51G1, (C) HYD1, (D) HYD2, and (E) 3βHSD/D amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp) are shown. The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

The second enzyme, CYP51G1, a cytochrome P450 (CYP) monooxygenase of the CYP51G gene family, is highly conserved in the animal, fungal, and plant kingdoms. However, higher plants use obtusifoliol to produce the precursors of BRs and structural sterols, whereas animals and fungi use lanosterol to synthesize cholesterol and ergosterol, respectively (Debeljak et al., 2003; Geisler et al., 2013; Kim et al., 2005b; O'Brien et al., 2005). In contrast to the single copy of CYP51 in yeast and mammals, some plants have several functional CYP51s owing to duplication events (Lepesheva and Waterman, 2007; O'Brien et al., 2005). Our phylogenetic analysis (Figure 2B) indicated the presence of four canonical CYP51 genes in maize; three in Setaria; two in soybean; and one in rice, Brachypodium, and Arabidopsis. Previous studies have indicated that the Arabidopsis and rice genomes contain other CYP51 genes, but only one is a functional CYP51G1 (Søren et al., 2011). In rice, the CYP51 family has 12 members—three OsCYP51Gs (1, 3, 4) and nine OsCYP51Hs (1–9)—but only OsCYP51G1 directly encodes a functional obtusifoliol 14α-demethylase (Inagaki et al., 2011; Xia et al., 2015; Jiao et al., 2020).

Investigation of CYP51G1 expression patterns among and within species indicated that these genes are generally highly expressed in several tissues (Figure 2B). By contrast, low expression of maize Zm00001eb170560 and Zm00001eb170580 could imply functional diversification or reflect pseudogene status. Soybean CYP51G1s are expressed primarily in seedlings and roots, whereas Bradi4g25930 is expressed in young seeds and inflorescences. OsCYP51G1 shows the highest expression in shoots and roots of rice, whereas AtCYP51G1 shows the highest expression in young seeds and roots of Arabidopsis.

Functional characterization of CYP51G1 genes in Arabidopsis and rice indicated that CYP51G1s are involved in shoot and embryonic development, anther and pollen development, reproductive heading, seed production, phytosterol biosynthesis, and auxin–ethylene signaling regulation (Kim et al., 2005b; Jiao et al., 2020). In Arabidopsis, the Atcyp51g1 mutants are seedling lethal with reduced organ size and defective cell expansion (Kim et al., 2005b). The Atcyp51g1 mutants were insensitive to exogenous BR treatment and accumulated obtusifoliol but had decreased CR and sitosterol levels (Kim et al., 2005b). Rice loss-of-function mutants of OsCYP51G1 exhibited growth defects similar to those of Atcyp51g1 but with delayed flowering, abnormal anther development, and pollen sterility (Jiao et al., 2020). OsCYP51G1 overexpression lines had increased grain weight and more seeds per panicle, highlighting the potential of OsCYP51G1 for crop yield improvement (Jiao et al., 2020). OsCYP51G3 and OsCYP51H3 are phylogenetically distant from OsCYP51G1, and their mutants are similar and indistinguishable from those of Oscyp51g1, thus suggesting that OsCYP51G1, OsCYP51G3, and OsCYP51H3 function collectively to regulate phytosterol synthesis and plant development. However, double and triple mutants should be produced to address this claim, and there is a need to investigate the role of OsCYP51s in other species.

4α-methylergostatrienol to 24-methylenelophenol

The enzymatic reduction of 4α-methylergostatrienol to 4α-methyl-fecosterol and the isomerization of 4α-methyl-fecosterol to 24-methylenelophenol are catalyzed by sterol C-14 reductase (encoded by HYD2 in Arabidopsis) and C-8,7 sterol isomerase (encoded by HYD1), respectively (Jang et al., 2000; Schrick et al., 2000; Souter et al., 2002). C-14 sterol reductase and C-8,7 sterol isomerase are conserved across plants, fungi, and animals (Jang et al., 2000; Pierre, 2002; Souter et al., 2002; Pullen et al., 2010). Our phylogenetic analysis resulted in two separate trees: HYD1 and HYD2 (Figure 2C and 2D). Arabidopsis, Brachypodium, and rice contain a single copy of the HYD1 gene, whereas other species contain two to four gene copies due to duplication events. HYD2 genes, on the other hand, are strictly single or two copy, with only intra-specific duplications. Despite these evolutionary disparities, HYD1 and HYD2 family members exhibit some similar expression patterns, suggesting some functional conservation between gene families and across species.

Functional roles of HYD1 and HYD2 in plant growth and development have been well characterized through mutant analysis in Arabidopsis. Loss-of-function mutants indicated that these genes are jointly implicated in embryonic cell patterning, vascular tissue development, auxin signaling, and ethylene signaling (Souter et al., 2002). Their single mutants have dwarf plant stature, defective apical-basal radial patterns, and abnormal cell division, with increased ethylene response, reduced expression of auxin-regulated genes (IAA1 and IAA2), and aberrant polar localization of auxin transporters (PIN1 and PIN2) (Jang et al., 2000; Souter et al., 2002; Short et al., 2018). Exogenous application of BR did not rescue the mutant growth defects, suggesting that AtHYDs may function in the production of other phytohormones such as auxin, ethylene, and BR (Pullen et al., 2010). HYD genes remain uncharacterized in monocots and soybeans and should be a focus for future research.

4α-carboxy-5α-cholesta-7,24-dien-3β-ol to episterol

The synthesis of episterol from 4α-carboxy-5α-cholesta-7,24-dien-3β-ol occurs in two successive reactions: the oxidative decarboxylation of 4α-carboxy-5α-cholesta-7,24-dien-3β-ol to episterone and then the reduction of episterone to episterol, which are catalyzed by 3β-HYDROXYSTEROID DEHYDROGENASE/C4 DEXCARBOXYLASE (3βHSD/D) (Rahier et al., 2006) and a 3-keto steroid reductase (Gachotte et al., 1999), respectively. The 3βHSD/Ds belong to the short-chain alcohol dehydrogenase/reductase family; they are conserved in eukaryotes and function in ergosterol, cholesterol, and phytosterol biosynthesis in humans, fungi, and plants, respectively (König et al., 2000; Simard et al., 2005; Helliwell et al., 2015). The 3βHSD/D protein is encoded by ERG26 in yeast and 3βHSD/D in Arabidopsis (Rahier et al., 2006). Conversion of episterone to episterol requires a 3-keto reductase as identified in yeast; however, no such homologs have been identified in plants (Gachotte et al., 1999; Desmond and Gribaldo, 2009).

To examine the evolutionary relationships among 3βHSD/Ds in plants, we constructed a gene tree that revealed that 3βHSD/D is encoded by several 3βHSD/D copies, depending on the species (Figure 2E). There are five copies in Setaria, four in soybean, three in maize, and two in Arabidopsis, Brachypodium, and rice. Functional characterization in Arabidopsis has implicated 3βHSD/Ds in several aspects of plant growth and development, including the regulation of auxin movement (Rahier et al., 2006). Neither the single mutants (At3βhsd/d1 or At3βhsd/d2) nor the double mutant (At3βhsd/d1; At3βhsd/d2) displayed obvious growth defects (Kim et al., 2012). However, a line overexpressing At3βHSD/D1 and At3βHSD/D2 had compact plant stature, wrinkled leaves, and a small inflorescence with impaired auxin localization. These growth defects were unaltered by BL treatment (Kim et al., 2012), although rescue with BR inhibitors was not attempted. No 3βHSD/Ds have been characterized in monocots or soybeans.

For the sterol biosynthesis genes discussed thus far, which produce episterone from squalene, no mutant phenotypes have been reported to be rescued by exogenous BR application. By contrast, mutants for the next set of enzymes have growth and developmental phenotypes that, as a rule, are rescued by exogenous BR application. Thus, the following steps are required exclusively for BR production and, together, have been referred to in the literature as the “brassinosteroid-dependent” segment of the BR biosynthesis pathway (Clouse, 2002).

Brassinosteroid-dependent biosynthesis pathway

Episterol to campesterol

After sequential reactions, including a C-4 demethylation catalyzed by SMO2 (discussed above) to produce episterol from 24-methylenelophenol, the beginning of the BR-dependent pathway is marked by the episterol to CR steps that occur in three successive reactions. First, C-5 desaturation of episterol to 5-dehydro-episterol is catalyzed by delta-7 sterol C-5 desaturase encoded by DWARF7/STEROL1 (DWF7/STE1) (Cheon et al., 2010; Silvestro et al., 2013). Second, reduction of sterol delta-5-dehydro-episterol to 24-methylene-cholesterol is catalyzed by a sterol delta-7 reductase encoded by DWARF5 (DWF5) (Choe et al., 2000; Silvestro et al., 2013; Inoue et al., 2017). Third, 24-methylene-cholesterol reduction to CR is catalyzed by a sterol C-24 reductase encoded by DWARF1 (DWF1) (Choe et al., 1999).

Parallel to the conversion of episterol to CR, these enzymes, DWF7, DWF5, and DWF1, also function in the sitosterol-stigmasterol biosynthetic pathway by catalyzing sitosterol synthesis from delta-7-avenasterol; their mutants have reduced CR and sitosterol levels with aberrant plant development and defective membrane structure (Clouse, 2002). Evolutionarily, DWF1, DWF5, and DWF7 (Figure 3A–3C) are highly conserved enzymes across the plant and fungal kingdoms (Sonawane et al., 2016), suggesting that their genes may have arisen early in eukaryotic evolution (Vriet et al., 2015). Interestingly, these genes split into separate clades with different outgroups, indicating that multiple rounds of duplication have resulted in enzymes with specific functions (Desmond and Gribaldo, 2009; Sonawane et al., 2016). The sizes of their gene families vary, with only DWF5 showing no evidence of gene duplication in the species reviewed here (Figure 3A–3C).Figure 3 Phylogeny and transcript abundance across different tissues of DWF7, DWF5, DWF1, and DET2 phytosterol and brassinosteroid biosynthetic enzyme families.

Maximum-approximate-likelihood phylogenetic trees of (A) DWF7, (B) DWF5, (C) DWF1, and (D) DET2 amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp) are shown. The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and the blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types. The expression boxes for ZmNA1 were left blank because there was not a gene annotation in the current maize genome version and the previous gene annotation version was therefore used for phylogenetic analysis.

DWF7 consists of duplicate pairs in Arabidopsis, soybean, and maize and only one gene in Brachypodium, rice, and Setaria (Figure 3A). These DWF genes are extensively expressed in several tissues. Functional analysis in Arabidopsis indicates that DWF7 is essential for plant development; however, their mutant phenotypes suggest that AtDWF7/STE1 and AtSTE1B function redundantly (Choe et al., 1999; Cheon et al., 2010). The DWF5 gene is present as a single copy across the species we surveyed (Figure 3B). Mutant analysis of AtDWF5 indicated its critical role in plant growth and development (Choe et al., 2000). The DWF5 gene has not been characterized in most species; thus, future functional studies will examine its functions and potential for use in crop improvement.

The DWF1 gene family (Figure 3C) consists of two gene copies in all surveyed species except Arabidopsis and rice. Available expression data suggest that, in each species, one copy is expressed more widely than the other, implying some sub-functionalization. The Atdwf1 and Atdwf5 mutants exhibited similar BR-deficient phenotypes and growth defects that were corrected by treatment with exogenous castasterone (CS) or BL (Choe et al., 1999; Inoue et al., 2017; Youn et al., 2018). Functional characterization showed that, because of defective sterol C-24 reductase, Osdwf1/brd2 and Zmna2 mutants were both semi-dwarfed with compressed internodes and erect dark-green short leaves; they exhibited few and feminized branches in maize and deformed panicles and small spikelets in rice (Hong et al., 2005; Best et al., 2016). Gmdwf1a and Gmdwf1b mutants exhibited a dwarf phenotype that was more severe in the double mutant, but only the single Gmdwf1a mutant had increased pod and seed production (Xiang et al., 2024).

In summary, DWF1, DWF5, and DWF7 have been implicated in the regulation of various aspects of plant growth and development, including reproductive development and grain yield (Hong et al., 2005; Best et al., 2016), root development (Choe et al., 1999; Youn et al., 2018), stomatal opening (Inoue et al., 2017), and leaf and internode expansion (Choe et al., 2000; Hong et al., 2005; Best et al., 2016). Consistent with the DWF1-DWF5-DWF7 reactions marking the onset of the BR-dependent pathway, and unlike defects in the upstream mutants described previously, the growth defects of dwf1, dwf5, and dwf7 mutants were rescued by treatment with exogenous BL or CS (Clouse, 2002).

Campesterol to brassinolide

BL is the most abundant BR in most species and has been considered the terminal product of the BR biosynthetic pathway (Choe, 2006; Wei and Li, 2016). However, recent studies have reported disparities in the bioactivity of BR compounds between monocots and dicots (Kim et al., 2008). For instance, in dicots, both BL and its precursor CS are detected in abundance, but BL is the most active end product of the pathway (Choe, 2006; Wei and Li, 2016). Conversely, CS is the most active and terminal BR in rice (Kim et al., 2008). By inference, CS may be the most active BR in other monocots; however, tests of this hypothesis have not been reported in the literature.

In overview, plants use CR to synthesize CS through either of two interconnected pathways, termed the campestanol (CN)-independent and CN-dependent pathways (Wei and Li, 2016; Ohnishi, 2018). In the CN-independent pathway, CR is converted to 22α-hydroxy-campesterol (22-OHCR), 22α-hydroxy-campest-4-en-3-one (22-OH-4-en-3-one), 22α-hydroxy-5α-campestan-en-3-one (22-OH-3-one), and then 3-epi-6-deoxoCT, which is converted to 6-deoxo-TY and converges with the late C-6 oxidation pathway (Ohnishi et al., 2012; Vriet et al., 2013; Ohnishi, 2018). The CN-dependent pathway consists of two interconnected parallel routes, the early and late C-6 oxidation pathways (Ohnishi, 2018). In the early C-6 oxidation pathway , CN is oxidized to 6-oxocampestanol (6-oxoCN), and 6-oxoCN is then converted to CS through cathasterone (CT), teasterone (TE), 3-dehydroteaserone (3DT), and typhasterol (TY) (Ohnishi et al., 2012; Ohnishi, 2018). In the late C-6 oxidation pathway, CN is hydroxylated to 6-deoxocathasterone (6-deoxo-CT), and 6-deoxoCT is oxidized to 6-deoxoteasterone (6-deoxoTE), 3-dehydro-6-deoxoteasterone (6-deoxo3DT), 6-deoxotyphasterol (6-deoxo-TY), 6-deoxocastasterone (6-deoxoCS), and finally CS (Ohnishi et al., 2012; Ohnishi, 2018). Through these steps, all highly conserved in the plant kingdom, the enzymatic conversion of CR to CS involves reduction, hydroxylation, and oxidation reactions catalyzed by one sterol 5α-reductase and several CYPs. The CYP enzymes that catalyze oxidation and hydroxylation reactions in BR biosynthesis include CYP90A1, CYP90B1, CYP90C, CYP90D, CYP85A, and CYP724 (Ohnishi et al., 2006b; Ohnishi, 2018).

Sterol 5α-reductase

The aforementioned sterol 5α-reductase is encoded by DEETIOLATED2/NANA1 (DET2/NA1) and catalyzes the C-5 reduction of campest-4-en-3-one (4-en-3-one), 22-OH-4-en-3-one, and 22,23-diOH-4-en-3-one to yield 5α-campestan-3-one (3-one), 22-OH-3-one, and 6-deoxo3DT, respectively (Ohnishi et al., 2012; Ohnishi, 2018; Wei and Li, 2020). In all species examined here, except soybean, DET2/NA1 is encoded by a single gene (Figure 3D). DET2/NA1 has been well characterized only in Arabidopsis (AtDET2) and maize (ZmNA1) (Chory et al., 1991; Choe et al., 1999; Hartwig et al., 2011). The Atdet2 and Zmna1 mutants exhibited growth and developmental defects similar to those of Atdwf1 and Zmna2 (Noguchi et al., 1999; Hartwig et al., 2011). However, the pleiotropic phenotypes of Atdet2 and Zmna1 were associated with a defective 5α-reductase that accumulates 4-en-3-one substrates downstream of AtDWF1/ZmNA2 (Noguchi et al., 1999; Hartwig et al., 2011).

C-22 hydroxylase

CYP90B/DWF4 and CYP724A/D11 catalyze the C-22 hydroxylation of CR, 4-en-3-one, 3-one, or CN to yield 22-OHCR, 22-OH-4-en-3-one, 22-OH-3-one, or 6-DeoxoCT, respectively (Figure 4; Ohnishi, 2018). Our phylogenetic analysis indicated that CYP90B/DWF4 and CYP724A/D11 assort into two clustered subclades (Ohnishi et al., 2006a; Ohnishi, 2018). CYP90B/DWF4 and CYP724A/D11 are single-copy genes in all species examined in this review, with the exception of soybean, which has three copies of CYP90B/DWF4 (Figure 4). CYP90B/DWF4 and CYP724A/D11 catalyze the C-22 hydroxylation of BRs. For instance, Arabidopsis Atdwf4 mutants exhibited growth defects indistinguishable from those of Atdet2 or Atdwf1, and overexpression of D11 (AtCYP724A1) complemented Atdwf4 mutants (Choe et al., 1998; Zhang et al., 2012a). In rice, both Osdwf4 and Osd11 loss-of-function mutants displayed mild phenotypic defects such as semi-dwarfed stature with erect leaves and small round grains, and the double mutants exhibited enhanced growth defects relative to either single mutant (Sakamoto et al., 2006). In Setaria, loss-of-function mutants of the CYP724A/D11 homolog Svbsl1 displayed phenotypes similar to those of Osdwf4 and Osd11 mutants (Yang et al., 2018). Mutants of ZmD11 have not been reported, but its gene function has been tested in other species; overexpression of ZmD11 and ZmDWF4 restored the growth of Osd11 and Atdwf4 mutants, respectively, suggesting that the functions of DWF4/CYP90B and CYP724A/D11 are conserved across plant species (Liu et al., 2007; Sun et al., 2021). Overexpression of ZmD11 also affected grain size and quality in both rice and maize (Sun et al., 2021).Figure 4 Phylogeny and transcript abundance across different tissues of the CYP450 enzyme family involved in multiple steps of brassinosteroid biosynthesis.

Maximum-approximate-likelihood phylogenetic tree of brassinosteroid biosynthetic CYP450 amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp). The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

C-23 hydroxylase

The C-23 hydroxylation of 22-hydroxylated BRs to yield 23-hydroxylated BRs is controlled by ROTUNDIFOLIA3 (ROT3)/CYP90C and CYP90Ds, which function redundantly as C-23 hydroxylases (Ohnishi et al., 2006b). ROT3/CYP90C and CYP90Ds predominantly convert 6-deoxoTE to 3-dehydro-6-deoxoTE, TE to 3-dehydroTE, 3-epi-6-deoxoCT to 6-deoxoTY, 22-OH-4-en-3-one to 3-dehydro-6-deoxoTE, 6-deoxoCT to 6-deoxoTE, and 6-deoxoTE to 3-dehydro-6-deoxoTE (Sakamoto et al., 2012; Ohnishi, 2018). Consistent with previous studies (Rizal et al., 2015), our phylogenetic analysis (Figure 4) indicated that ROT3/CYP90C1 and CYP90D1 are restricted to dicots, whereas CYP90D2 and CYP90D3 are specific to monocots owing to a duplication event that occurred after the monocot–dicot divergence and before the evolution of grasses. In Arabidopsis, CYP90C and CYP90D enzymes are encoded by ROT3/CYP90C1 and CYP90D1, respectively (Kim et al., 2005a; Ohnishi et al., 2006b). Mutant studies indicated that ROT3/CYP90C1 and CYP90D1 encode functionally redundant C-23 hydroxylases. Atrot3 or Atcyp90d1 mutants displayed weak to no obvious BR-deficient phenotypes, but Atrot3;cyp90d1 double mutants displayed extreme dwarf phenotypes indistinguishable from those of Atdet2 and Atdwf4 mutants (Kim et al., 2005a; Ohnishi et al., 2006b). In monocots, the C-23 hydroxylation reactions are catalyzed by CYP90D2 and CYP90D3, encoded by a duplicate gene pair (Hong et al., 2003; Sakamoto et al., 2012; Rizal et al., 2015). The CYP90D2 and CYP90D3 proteins have overlapping functions; for instance, CYP90D2 loss-of-function mutants in rice and sorghum have weak growth defects compared with those of other BR-deficient mutants (Hong et al., 2003; Sakamoto et al., 2012; Rizal et al., 2015), perhaps because of partial complementation by CYP90D3. Similarly, single mutants of maize genes that are orthologs of OsCYP90D2 and OsCYP90D3 exhibit weak to no obvious BR-related growth defects, but double mutants are extremely dwarfed and indistinguishable from Zmna1 and Zmna2 mutants (B.Z. and E.V., unpublished data). These data further confirm that ZmCYP90D2 and ZmCYP90D3 function redundantly as C-23 hydroxylases.

C-3 oxidases

CYP90As function as C-3 oxidases and mediate the C-3 oxidation of CN, 22-OHCR, and (22R,23R)-22,23-dihydroxycampesterol to produce 4-en-3-one, 22-OH-4-en-3-one, and (22R,23R)-22,23-dihydroxy-campest-4-en-3-one, respectively (Ohnishi et al., 2012). CYP90A in Arabidopsis is encoded by CONSTITUTIVE PHOTOMORPHOGENIC DWARFISM (CPD) (Ohnishi et al., 2012), and CPD gene copy numbers vary. Arabidopsis and Brachypodium have a single CPD gene, whereas soybean has four and maize, Setaria, and rice have two because of gene duplication (Figure 4). In Arabidopsis, AtCPD has been extensively characterized through mutant screens and metabolite profiling, and Atcpd mutants accumulated 22-OHCR and exhibited extreme growth defects similar to those reported for Atdwf1 and Atdet2 mutants (Szekeres et al., 1996; Ohnishi et al., 2012). Recently, OsCPD loss-of-function mutants (Oscpd1 and Oscpd2) were reported; neither single mutant has obvious growth defects, but the double mutant oscpd1;oscpd2 displays multiple developmental defects similar to those of Osdwf4; Osd11 double mutants (Zhan et al., 2022), indicating functional redundancy. Similarly, single mutants of maize orthologs of OsCPD1 and OsCPD2 have no obvious growth defects, but double mutants have extreme growth defects (B.Z. and E.V., unpublished data).

C-6 oxidases

CYP85A/BR-C-6 oxidase catalyzes the oxidation of CS to BL. CYP85A also mediates several intermediate reactions, including the enzymatic conversion of 6-deoxoTE, 6-deoxo3DT, 6-deoxo-TY, and 6-deoxoCS to TE, 3DT, TY, and CS. The CYP85A/BR6OX genes encode BR-C-6 oxidase, and their copy numbers vary between dicots and monocots. Monocots have a single gene copy (dubbed CYP85A1), whereas Arabidopsis contains two gene copies (CYP85A1 and CYP85A2), and soybean has five CYP85As (Jager et al., 2007; Kim et al., 2008). Unlike in Setaria, soybean, and Brachypodium, CYP85A loss-of-function mutants have been well characterized in Arabidopsis, maize, and rice. Mutant studies in Arabidopsis indicated that AtCYP85A1/AtBR6OX1 and AtCYP85A2/AtBR6OX2 catalyze similar reactions and function collectively. For instance, Atbr6ox1 and Atbr6ox2 mutants have mild to no obvious growth defects, and their double-mutant growth defects were enhanced and were restored by application of exogenous BL and CS, emphasizing that BL and/or CS activity is required for plant growth and development (Kim et al., 2005c; Kwon et al., 2005).

ZmBRD1 and OsBRD1 loss-of-function mutants in maize and rice, respectively, showed various pleiotropic vegetative and reproductive defects, highlighting the essentiality of CYP85A1 in BR biosynthesis and plant development (Mori et al., 2002; Makarevitch et al., 2012). Zmbrd1 mutants were indistinguishable from Zmna1 and Zmna2 mutants (Baluška et al., 2001; Makarevitch et al., 2012; Castorina et al., 2018). These mutants also exhibited BR-deficiency-related defects at the seedling level that were partially rescued by exogenous BL (Makarevitch et al., 2012). Plant growth and developmental defects of Osbrd1 mutants were similar to those reported for Zmbrd1 mutants (Hong et al., 2002; Makarevitch et al., 2012). Despite the partial rescue of Zmbrd1 and Osbrd1 phenotypic defects by exogenous BL, recent studies have shown that monocots likely use CS instead of BL as the primary endogenous bioactive BR. CS was shown to be the end product of BR biosynthesis in rice, and it was noted that rice (and every monocot) lacks a CYP85A2 that may more specifically catalyze the conversion of CS to BL (Kim et al., 2008; Roh et al., 2020). There is a need to carefully profile metabolite accumulation levels and quantify the extent of growth rescue by applying BL and CS to CYP85A loss-of-function mutants in order to analyze CS and BL as end products of BR biosynthesis.

Brassinosteroid movement

Local transport is necessary for bioactive BRs to exit the cell and bind to the BR receptor anchored in the cell membrane. How BRs move through the intracellular environment from the endoplasmic reticulum membrane to the plasma membrane for export is not yet known. It has been hypothesized that movement could occur either through the creation of BR conjugates or through the binding of BRs to protein complexes, which would reduce their hydrophobicity and facilitate their movement (Fujioka and Yokota, 2003; Marković-Housley et al., 2003; Sasse, 2003; Choe, 2006). Once BRs reach the cell membrane, they need to cross the lipid bilayer by an active, passive, or combined mechanism to exit the cell. Symplastic cell-to-cell transport of BR precursors via plasmodesmata has been demonstrated (Nolan et al., 2020). A recent publication also showed that the ATP binding cassette transporters ABCB19 and ABCB1 could transport BRs across the plasma membrane (Ying et al., 2024). Given that BRs can feedback regulate biosynthesis and signaling components (Wei and Li, 2020), BRs can likely bind to receptors of the same cell that synthesized them. Apoplastic movement of BRs should also make it possible for BRs to influence nearby cells in a given tissue or organ.

Long-distance transport of BRs has been investigated in a few species, including tomatoes and peas (Symons et al., 2008). Exogenous application of BRs to the roots can recover biosynthetic mutant phenotypes, as evident in Arabidopsis shoots (Choe et al., 1998). However, grafting experiments in tomato and pea showed that grafting wild-type rootstocks to mutant scions could not recover the dwarf shoot phenotype, indicating that BRs synthesized in vivo were not transported acropetally (Symons and Reid, 2004; Montoya et al., 2005). Furthermore, grafting wild-type scions to mutant rootstocks did not increase endogenous levels of BRs in the root stock, indicating a lack of long-distance basipetal transport (Symons and Reid, 2004; Montoya et al., 2005). At present, there is no evidence that endogenous BRs are transported long distances throughout the plant.

Brassinosteroid catabolism and homeostasis

BR homeostasis comprises a complex interplay among the spatiotemporal distribution of active BR compound(s); the perception, signaling, and response to these BRs; and inputs from the genotype and environment. Given the apparent lack of long-distance active transport of BRs (Symons et al., 2008), the tissue composition of BRs is largely determined by local metabolism, consisting of biosynthesis, as discussed above, and catabolic processes such as conjugation, modification, and degradation. Catabolism of BL and CS in vivo was first documented by the discovery in Arabidopsis of the C26 hydroxylase PHYB ACTIVATION-TAGGED SUPPRESSOR1 (BAS1), which, when overexpressed, mimicked BR biosynthetic and response mutants and caused reduced levels of BRs (Neff et al., 1999). BAS1 is a single-copy gene in Arabidopsis, maize, Setaria, and Brachypodium, and there are three copies in soybean (Supplemental Figure 3).

Genetically related C26 hydroxylases have since been analyzed in many plant species. Several different conjugating and modifying activities that act either directly on the predominant bioactive compounds BL and CS and/or on biosynthetic intermediates have now been documented, including glycosyltransferases, a reductase, acyltransferases, and sulfotransferases (Vriet et al., 2013; Wei and Li, 2020). In addition to biochemical evidence, in planta genetic evidence has demonstrated that, for many catabolic genes, overexpression mimics BR-deficient phenotypes, whereas loss-of-function mutants show organ elongation or other BR-responsive phenotypes (Neff et al., 1999; Turk et al., 2005; Thornton et al., 2010; Schneider et al., 2012; Zhuang et al., 2022). Mechanisms that regulate BR catabolic genes during plant development and in response to external cues have also begun to be characterized (Wei and Li, 2020). A gene with sequence similarity to AtBAS1, OsBRASSINOSTEROID-DEFICIENT DWARF3, was ectopically expressed in rice panicles, where it increased panicle branching and yield without affecting seed size (Zhang et al., 2024); these data suggest a target for future crop improvement in, e.g., wheat and Setaria, but likely not in maize, where the ear is unbranched.

Brassinosteroid perception

BRASSINOSTEROID INSENSTIVE1/BRASSINOSTEROID INSENSITIVE-LIKE (BRI1/BRL)

BRs are perceived at the plasma membrane through binding of bioactive BRs to the BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRASSINOSTEROID INSENSITIVE1-LIKE (BRL) receptor kinases (Clouse et al., 1996; Li and Chory, 1997; Cano-Delgado et al., 2004) (Supplemental Figure 4). There are four BRI1/BRL family members in Arabidopsis, rice, Brachypodium, and Setaria, five in maize, and six in soybean (Figure 5A). Most duplications and divergences in this family predate the split between monocots and eudicots, with the exception of BRI1 homologs. Single BRI1 orthologs are present in rice and Setaria, but recent genome duplications in maize, Brachypodium, and soybean have given rise to an additional BRI1 gene via duplication of the closest homolog of AtBRI1, followed by retention of both copies. The second Brachypodium BRI1 gene is likely a pseudogene, as it encodes variant amino acids at several otherwise well-conserved residues in the kinase domain. Across species, BRI1 genes are more highly expressed than BRL gene family member(s), with the highest BRI1 expression in developing inflorescences and floral tissues. BRL genes are most highly expressed in root tissue, except for OsBRL2, whose expression is highest in young inflorescences. The similar BRI1/BRL expression patterns across species suggest an ancient, conserved sub-functionalization among monocots and eudicots.Figure 5 Phylogeny and transcript abundance across different tissues of BRI1 and BAK1 families involved in brassinosteroid perception.

Maximum-approximate-likelihood phylogenetic trees of (A) BRI1 and (B) BAK amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp) are shown. The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

The first Atbri1 mutants were discovered in mutant screens for loci insensitive to epi-BL treatment (Clouse et al., 1996; Li and Chory, 1997). In addition to extracellular LRR and ID domains essential for BR binding and perception at the cell membrane (Kinoshita et al., 2005; She et al., 2011), BRI1 proteins contain a signal peptide, a putative leucine-zipper motif, a transmembrane domain, and a cytoplasmic kinase domain that all contribute to BRI function in perceiving BRs and initializing the phosphorylation cascade necessary for BR signaling (Li and Chory, 1997). The BRL gene products, AtBRL1 and AtBRL3, were also shown to bind BL with high affinity. Overexpressed AtBRL2 could not bind BL (Cano-Delgado et al., 2004; Zhou et al., 2004). When BRs are not present, BRI1 function is inhibited by BRI1 KINASE INHIBITOR1 (BKI1) (Supplemental Figure 5A; Wang and Chory, 2006; Wang et al., 2014a). In addition, a dominant mutant of AtBRI1 SUPPRESSOR1 (AtBRS1) was able to suppress some phenotypes of Atbri1-5, but no interaction partners of AtBRS1 have been identified (Supplemental Figure 5B; Li et al., 2001b). Transgenic expression of GmBRI1A in the Atbri1-5 mutant background (Wang et al., 2014b) and overexpression of GmBRI1B in the Atbri1-6 mutant background (Peng et al., 2016) were able to rescue the phenotypes of the Arabidopsis mutants, indicating conservation of function across species.

To initially characterize the BRI1/BRL gene family in maize, RNA-interference (RNAi) transgenic plants were created to knockdown transcript levels (Kir et al., 2015). The RNAi constructs were designed to target ZmBRI1A and ZmBRI1B, but transcript levels were reduced for all five BRI1/BRL homologs. The ZmBRI1-RNAi lines were moderately dwarfed owing to inhibited elongation of all internodes, especially the upper internodes between the ear and tassel. Leaf blade and leaf sheath length were also affected, but leaf blade width was not (Kir et al., 2015). Interestingly, despite the knockdown of all BRI1/BRL homologs, none of the transgenic maize lines obtained by the transgenic RNAi approach showed the extreme dwarfism of bri1 single mutants from Arabidopsis or rice, suggesting a need for mutant analysis in maize.

The OsD61 gene encodes OsBRI1 in rice, and the Osd61 mutant phenotype closely resembles that of the biosynthetic mutant Osbrd1-1 (Nakamura et al., 2006). The severe Osd61-4 allele has a mutation in the kinase domain of OsBRI1, demonstrating that OsD61 is necessary for regulation of BR signaling (Zhao et al., 2013). Osd61 mutant plants were severely dwarfed with abnormal leaves and were sterile owing to a lack of flowers. RT–PCR analysis showed that OsBRI1 was expressed primarily in shoots, whereas OsBRL1 and OsBRL3 were expressed in roots, as observed in our expression analysis (Figure 5A). CS levels were close to 30 times higher in Osd61 mutant shoot tissue than in that of its wild-type siblings but were only 1.5 times higher in root tissue. The reduction in mutant root length was also much milder compared with that of Osbrd1-1 (Nakamura et al., 2006). These results suggest that other receptors, such as OsBRL1 and OsBRL3, may be sufficient or may compensate for the lack of OsD61 function in root tissue.

Transgenic RNAi lines have also been developed to target and lower the expression of BdBRI1 (Feng et al., 2015). These RNAi lines exhibited stunted growth but not extreme dwarfism, as observed in maize. The BdBRI1 RNAi lines were also more drought tolerant and exhibited altered expression of drought-responsive genes under drought conditions (Feng et al., 2015), highlighting a potential applied use of BR mutants for drought tolerance across species. Surprisingly, overexpression of BdBRI1 in the Atbri1-5 background did not recover the mutant phenotype (Corvalán and Choe, 2017). The function of BRI1/BRL proteins has not been studied in Setaria.

BRI1-ASSOCIATED RECEPTOR KINASE (BAK1)

The BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) gene encodes an LRR receptor-like kinase similar to BRI1 that belongs to the somatic embryogenesis receptor kinase (SERK) family. Four other Arabidopsis genes show high sequence similarity to AtBAK1; all five genes are highly expressed in developing inflorescences, and AtBAK1 is also highly expressed in seedling roots (Figure 5B). The four BAK1-like genes in maize also show high expression in developing inflorescences. Phylogenetic analysis suggests that, in contrast to the ancient conservation of BRI1 genes, the BAK1 gene family underwent independent expansions in eudicots and grass species (Figure 5B). The ZmSERK2 and ZmSERK3 genes, whose expression pattern is similar to that of AtBAK1, both show high expression in seedling roots, possibly indicating their redundant function in maize. There are eight BAK1-like genes in soybean. In rice, two genes encode polypeptides with sequence similarity to AtBAK1/SERKs. Three additional genes (LOC_Os08g07890, LOC_Os06g12120, and LOC_Os08g07890) show phylogenetic and gene structural features that suggest altered functions (Man et al., 2020), and they were left out of our phylogenetic and expression analyses. OsBAK1 expression is higher in developing inflorescences, whereas OsSERK2 expression is higher in seedling tissues, suggesting some functional diversification in the regulation of BR signaling. Setaria has only three BAK1-like genes, and SvSERK1 and SvSERK4 have higher average expression than SvBAK1. The three BAK1-like genes in Brachypodium show expression profiles similar to those of genes in their respective subclades.

The AtBAK1 gene was discovered by screening for suppressors of the Atbri1 mutant. Dominant Atbak1-1D mutants suppressed the short inflorescence, shortened petiole, and curled lamina phenotypes of Atbri1-5. The loss-of-function mutant Atbak1-1 was semi-dwarfed, with reduced sensitivity to BL-induced root length inhibition. BAK1 localizes to the plasma membrane and physically interacts with BRI1 in vitro and in vivo. When BRs are absent, BRI1 and BAK1 are not phosphorylated and do not interact. When BRs bind to BRI1, BRI1 associates with BAK1, and both become auto- and/or trans-phosphorylated, initiating an intracellular phosphorylation cascade to activate BR signaling and response (Li et al., 2002). AtSERK1 and AtBAK1 are functionally redundant in BR signaling. AtBAK1 also acts as a co-receptor to regulate plant innate immunity (Albrecht et al., 2008); however, some immune responses are regulated by BRs independently of AtBAK1 (Albrecht et al., 2012; Belkhadir et al., 2012).

Loss-of-function Atbak1 mutants have short primary roots, and double or triple mutants with loss-of-function Atserk1 and/or Atserk2 alleles showed further reductions in primary root length (Du et al., 2012; Ou et al., 2022). Root growth and development have been shown to be regulated by AtROOT MERISTEM GROWTH FACTOR1 (RGF)/GLOVEN(GLV)/CLE-like peptides (Fernandez et al., 2013), a class of small peptide hormones that are perceived by the AtRGF1 INSENSITIVE (RGI) leucine-rich repeat receptor-like kinase (Ou et al., 2016; Shinohara et al., 2016; Song et al., 2016). These RGIs act as co-receptors with AtBAK1 and other AtSERKs to bind RGFs and regulate the root meristem cell niche, lateral root development, and root gravitropism (Ou et al., 2022). The regulation of root growth by RGF/RGI has not been directly linked to BRs, suggesting that AtBAK1/SERK proteins regulate root growth and development through both BR-dependent and BR-independent pathways.

Arabidopsis BAK1-INTERACTING RECEPTOR-LIKE KINASE2 (BIR2) and BIR3 were also shown to interact with AtBAK1 and AtBRI1 in vivo to regulate their function (Halter et al., 2014; Imkampe et al., 2017). AtBIR3 stabilizes AtBAK1 and prevents it from associating with ligand-binding receptors to form a receptor complex (Imkampe et al., 2017). Also independently of BRs, AtSERK1 and AtSERK2 were responsible for male microsporogenesis. In soybean, GmBAK1 was shown to form a complex with GmFLAGELLIN-SENSITIVE2 to perceive flagella from Ralstonia solanacearum, apparently independently of BRs (Wei et al., 2020).

Maize BAK1/SERK genes have not yet been characterized through mutant analyses. Transcriptomic studies on somatic embryogenesis in maize demonstrated upregulation of Zm00001eb414790, but neither ZmSERK1 nor ZmSERK2 nor ZmSERK3 were differentially regulated during this process (Ding et al., 2020). This surprising result may suggest some diversification between Arabidopsis and maize in the function of BAK1/SERK genes (Salaj et al., 2008).

To identify the function of BAK1/SERK genes in rice growth and development, RNAi knockdown lines were developed. Multiple constructs targeted OsBAK1, OsSERK2, and the additional SERK-like gene OsORK1 (LOC_Os08g07890). Severe phenotype events with no detectable expression of OsBAK1 were dwarfed and insensitive to BL-induced laminar leaf bending (Park et al., 2011). Overexpression in Arabidopsis of OsBAK1 and OsSERK2, but not of OsSERK3 (LOC_Os06g12120) or OsSERK4 (LOC_Os02g18320), complemented the mutant phenotypes of a weak Atbri1 allele, indicating conservation of BAK1/SERK1 molecular function across species (Li et al., 2009a) despite their phylogenetic history (Figure 5B). The Osserk2 mutant has been cloned and showed an altered response to BL treatment (Dong et al., 2020). Thus, OsBAK1 and OsSERK2 genes are most closely related to AtBAK1 and act redundantly in the regulation of BR signaling. Future work is needed to investigate the functions of BAK1/SERK genes in Setaria, Brachypodium, and soybean.

Brassinosteroid signal transduction

Regulation of brassinosteroid transcriptional response

BRASSINOSTEROID INSENSTIVE2/GLYCOGEN SYNTHASE KINASE3 /SHAGGY-LIKE PROTEIN KINASE1 (BIN2/GSK)

The AtBIN2 gene encodes a GLYCOGEN SYNTHASE KINASE3 (GSK3)-like protein that acts as a negative regulator of BR signaling. BRASSINOSTEROID INSENSITIVE2 (BIN2) activity is directly regulated by CDG1 and BSU1 (Supplemental Figure 5C) (Kim et al., 2009). The GSK3 subfamily has 10 genes in Arabidopsis that cluster into five separate subclades (Dornelas et al., 1998; Youn and Kim, 2015). Soybean has 22 GSK3-like genes, maize has 13, rice has nine, Brachypodium has eight, and Setaria has eight. One of the Arabidopsis and soybean subclades, which contains AtBIN2 along with AtBIL1 and AtBIL2, clusters in the gene tree (Figure 6) as a sister group to five maize genes, four rice genes, three Brachypodium genes, and three Setaria genes. The grass genes resolve into two additional sister subclades, and maize, rice, and Setaria are represented in all three subclades, whereas Brachypodium is represented in only one (Figure 6). Transcripts of AtBIN2, AtBIL1, and AtBIL2 are most highly expressed in young inflorescences and carpels and show high expression in seedling shoots, roots, and young seeds. The soybean, maize, rice, Brachypodium, and Setaria genes in this subclade also have their highest transcript expression levels in inflorescences, with the exception of two soybean genes (Glyma.12G212000 and Glyma.13G228100) whose highest expression is in developing seeds (Figure 6).Figure 6 Phylogeny and transcript abundance across different tissues of the BIN2/GSK family involved in brassinosteroid signal transduction.

Maximum-approximate-likelihood phylogenetic tree of BIN2/GSK amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp). The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow color indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

The AtSK13 subclade is similarly complex and includes two additional Arabidopsis genes, AtSK11 and AtSK12, and several soybean, Setaria, Brachypodium, maize, and rice genes. Increased copy numbers in maize and soybean likely reflect a duplication of this gene specific to both species. Like AtBIN2/BIL transcripts, AtSK11 and AtSK12 transcripts accumulate across tissues, showing their highest expression in inflorescences and carpels. These genes also have high transcript abundance in seedling tissues and accumulate in young seeds (Figure 6).

AtSK31 and AtSK32 are most highly expressed in anthers, with AtSK31 having the highest average fragments per kilobase of exon per million mapped fragments (FPKM) values across all tissues. Related paralogs in other species also show the highest expression in inflorescences (Figure 6). Arabidopsis AtSK41 and AtSK42 genes form a final subclade that includes a single gene each from maize, rice, Brachypodium, and Setaria, but there are two soybean genes, as in Arabidopsis, showing a diversification between eudicots and monocots. AtSK41 and AtSK42 have relatively uniform transcript abundance across all tissues, with their lowest abundance in developing seeds and embryos. Related maize, rice, Brachypodium, and Setaria genes show similar transcript abundance across tissues compared with the Arabidopsis genes, although the grass genes have higher expression in developing embryos (Figure 6).

BIN2 was first characterized in Arabidopsis by identification of the semi-dominant mutant Atbin2, which resembled BR-deficient mutants and was hyposensitive to BL treatment for root seedling elongation phenotypes (Li et al., 2001a). The negative regulation of BR responses by BIN2 was further supported by observations that overexpression of BdBIN2 produced a dwarf phenotype in Arabidopsis (Corvalán and Choe, 2017).

Atbin2 mutants were also hypersensitive to ABA treatment, whereas their roots were significantly shorter than those of wild-type plants (Clouse et al., 1996; Ephritikhine et al., 1999), suggesting crosstalk between BR and ABA in the regulation of root growth in Arabidopsis. In investigations of BR and auxin crosstalk, root growth of Atbin2 mutants was mildly hyposensitive to synthetic auxin treatment (Choe et al., 2002) but not to natural auxin treatment (Li et al., 2001a). The infertility phenotype of dominant Atbin2 mutants could be partially rescued by exogenous application of the synthetic auxin naphthaleneacetic acid (Li et al., 2020b), suggesting a complex interaction between auxin and BRs in the regulation of plant development. A key function of the BIN2 protein kinase is phosphorylation of BRI1-EMS-SUPPRESSOR1 (BES1)/BRASSINAZOLE-RESISTANT1 (BZR1) transcription factors (He et al., 2002). Dominant Atbin2 alleles contain mutations in the TREE motif, part of the BIN2 catalytic domain, which result in higher kinase activity and higher levels of endogenous BRs (Choe et al., 2002; Li and Nam, 2002; Yan et al., 2009).

AtBIN2 has also been shown to regulate the function of other proteins and pathways in BR signaling in addition to those downstream of BES1/BZR1 transcription factors. For example, BIN2 regulates cell elongation independently of BES1/BZR1 transcription factor–coupled pathways by phosphorylating AUXIN RESPONSE FACTOR2 (ARF2), PHYTOCHROME INTERACTING FACTOR4 (PIF4), and CESTA transcription factors (Vert and Chory, 2006; Bernardo-Garcia et al., 2014; Khan et al., 2014). Regulation of root development by AtBIN2 has been shown by its ability to phosphorylate and regulate the function of AtARF7, AtTRANSPARENT TESTA GLABRA1 (AtTTG1), and AtENHANCER OF GLABRA3 (AtEGL3) (Cheng et al., 2014; Cho et al., 2014). AtBIN2 can also negatively regulate cellulose synthesis by directly phosphorylating AtCELLULOSE SYNTHASE A1 and inhibiting its function, and dominant Atbin2 mutants have lower cellulose content (Sanchez-Rodriguez et al., 2017). AtBIN2 is also involved in drought response, in which phosphorylation of AtRD26 results in increased activation and transcriptional regulation of drought-responsive genes (Jiang et al., 2019). AtBIN2 has an additional role in directly regulating the balance between salt stress and growth recovery in Arabidopsis (Li et al., 2020a). These findings were supported by overexpression of GmBIN2 in Arabidopsis and soybean hairy roots, which resulted in greater drought and salt tolerance (Wang et al., 2018). Taken together, these studies suggest that the activity of BR signaling through BIN2 can regulate multiple plant growth and stress responses independently of transcription factors in the canonical BR-response pathway.

To test the possible redundancy between AtBIN2 and other GSK3 kinase genes in Arabidopsis, Atbin2 loss-of-function alleles were developed by mutagenizing plants containing a weak dominant Atbin2 allele and screening for intragenic suppressors that eliminated the dominant phenotype. Several loss-of-function Atbin2 alleles showed no obvious phenotypes compared with wild-type siblings (Yan et al., 2009), likely because of the redundancy of GSK3 proteins. However, the loss-of-function allele Atbin2-3 did partially recover the Atbri1-5 dwarf phenotype, and single mutants of Atbin2-3 were slightly hyposensitive to BL treatment for root and hypocotyl growth. T-DNA mutant lines of Atbil1 and Atbil2 were characterized to test the possible redundancy among GSK3 proteins. Single and double mutants between Atbil1 and Atbil2 exhibited no visible phenotypes under normal growth conditions, but, like Atbin2-3, they were less sensitive to BR and BRZ application, suggesting redundancy among these three genes. The single Atbin2, Atbil1, and Atbil2 loss-of-function mutants were also hyposensitive to ABA-induced root inhibition owing to their interaction with AtSNF1-RELATED KINASE2S (Cai et al., 2014). Beyond its role in BR signaling, AtBIL1 also functions in auxin and cytokinin signaling through phosphorylation of AtMONOPTEROS to transcriptionally activate negative regulators of cytokinin signaling and control proliferation of the vascular cambium (Han et al., 2018). The double and triple mutants of Atbil1 and/or Atbil2 with Atbri1-5 could not recover the Atbri1-5 dwarf phenotype. However, mutant combinations of Atbil1 and/or Atbil2 with Atbin2-3 and Atbri1 exhibited greatly enhanced suppression of the Atbri1-5 phenotype, indicating further redundancy of these loci in Arabidopsis. Interestingly, triple mutants of Atbin2-3, Atbil1, and Atbil2 were still responsive to BRs, suggesting that other GSK3s may also function in BR signaling (Yan et al., 2009).

Consistent with this hypothesis, overexpressed AtSK32 protein was shown to directly phosphorylate AtBES1/AtBZR1 transcription factors and regulate their transport into the nucleus (Kim et al., 2009; Rozhon et al., 2010). Furthermore, recessive mutations of Atsk32 result in reduced hypocotyl elongation and altered floral organ cell size (Claisse et al., 2007). The AtSK31 protein is a component of the CONSTITUTIVE PHOTOMORPHOGENIC9 (COP9) complex, which represses photomorphogenesis and induces skotomorphogenesis (Wei et al., 1994a; Chamovitz et al., 1996; Staub et al., 1996). Mutants of the COP9 complex, including Atsk31 (Wei et al., 1994b), have phenotypes that resemble those of BR-deficient mutants when grown in the dark, but no direct connection has been made between the COP9 complex and BRs (Karniol and Chamovitz, 2000).

The AtSK11 and AtSK12 proteins have also been shown to participate in BR signaling by physically interacting with BES1/BZR1 transcription factors in yeast two-hybrid and co-immunoprecipitation assays (Kim et al., 2009; Tang et al., 2011). Transgenic antisense constructs of AtSK11 and AtSK12 led to abnormal flower development with increased sepal, petal, and flower bud numbers and a greater number of floral meristems compared with wild-type siblings. Antisense plants also had altered gynoecium apical-basal patterning and a larger gynophore at the basal end of the gynoecium. However, the antisense transgenes did not affect flowering time or the pattern of cell division (Dornelas et al., 2000). By contrast, the AtSK12 protein was recently shown to regulate flowering by degrading CONSTANS via direct phosphorylation. Recessive mutants of Atsk12 flowered earlier and had fewer rosette leaves (Chen et al., 2020), suggesting that the antisense lines were knockdowns with residual AtSK function.

Among additional regulators of AtBIN2, the F box E3 ubiquitin ligase AtKINK SUPPRESSED IN BZR1-1D (AtKIB1) was also shown to regulate AtBIN2 function via ubiquitination and subsequent targeting for proteasomal degradation. AtKIB1 could also inhibit AtBIN2 by interfering with substrate access of other interacting proteins, including the AtBES1/AtBZR1 transcription factors. The AtKIB1 protein could physically interact with AtBIN2 as shown by in vivo co-immunoprecipitation experiments, and the C-terminal end of AtKIB1 was sufficient to interact with AtBIN2 in yeast two-hybrid assays (Zhu et al., 2017). The C-terminal end of AtKIB1 was also shown to physically interact with AtBIL1, AtBIL2, AtSK11, AtSK12, and AtSK13 in yeast two-hybrid assays (Zhu et al., 2017), further implicating these proteins in BR signaling and suggesting significant redundancy among these members of the GSK3 family. In rice, the U-box ubiquitin ligase OsTUD1 was shown to physically interact with OsGSK2, ubiquitinating it and inhibiting its function (Liu et al., 2023). The AtSK41 and AtSK42 proteins show high amino acid sequence similarity to AtBIN2 (Jonak and Hirt, 2002). However, their functions in plant development and/or BR signaling have not yet been characterized. AtSK42 transcript levels increased after sodium chloride and polyethylene glycol treatments, whereas AtSK41 transcription was unresponsive (Charrier et al., 2002).

The maize BIN2/GSK3 genes have been investigated through development of RNAi lines (Kir, 2015). These lines redundantly targeted all ZmBIN2/ZmGSK3-like transcripts using the full-length cDNA of Zm00001eb204920. This resulted in lower transcript accumulation for all loci tested in leaf tissue; however, Zm00001eb338190 and Zm00001eb417200 were not expressed in this tissue (Kir, 2015), and Zm00001eb20224460, Zm00001eb404640, and Zm00001eb021340 were not tested (Figure 6). Therefore, RNAi phenotypes could not be assigned to a particular locus. All RNAi events produced a reduction in plant height due to a relatively uniform reduction in internode length throughout development (Kir, 2015). These knockdown phenotypes are surprising given that combinations of BIN2/GSK3 mutant alleles in Arabidopsis did not show visible phenotypes (Yan et al., 2009) and thus underscore the necessity of developing higher-order mutant combinations in Arabidopsis to alleviate phenotypic masking due to functional redundancy of these homologs. The Zmbin2-RNAi lines flowered later than their wild-type siblings; their inflorescences were elongated and had a lower spikelet density with many barren tips, suggesting a reduction in spikelet pair initiation and/or development. Interestingly, Zmbin2-RNAi lines also showed maternally increased kernel number and embryo size (Kir, 2015). Similar to Arabidopsis mutants, Zmbin2-RNAi lines had reduced root growth (Yan et al., 2009). However, the Zmbin2-RNAi lines were hyposensitive to BL treatment (Kir, 2015), unlike Arabidopsis Atbin2-3 mutants, which were hypersensitive to the effects of BL on root growth (Yan et al., 2009).

The rice OsGSK1, OsGSK2, OsGSK3, and OsGSK4 genes have been well described. Development of overexpression, RNAi, and CRISPR-Cas9 lines has enabled comparison of their different contributions to the control of plant development. Overexpression of OsGSK2 had no obvious effect on plants, but overexpression of mutated forms with presumed hyperactive kinase activity resulted in strong BR-deficiency phenotypes, including reduced plant height, tillering, and growth of leaf sheathes and blades, as well as upright leaf angle, delayed flowering time, erect panicles, some sterility, and reduced grain size (Tong et al., 2012). RNAi lines targeting OsGSK2 had reduced transcript levels of OsGSK1, OsGSK2, OsGSK3, and OsGSK4. These RNAi lines showed no changes in plant height but did exhibit less upright leaves that were longer and narrower than those of wild-type siblings. These transgenic lines also showed increased grain length (Liu et al., 2021a; Tong et al., 2012). Osgsk2 single mutants and double mutants with Osgsk1, Osgsk3, or Osgsk4 were significantly taller and had more upright leaves than wild-type siblings. Triple mutants between Osgsk1, Osgsk2, and Osgsk3 exhibited no significant change in plant height but had a less upright leaf angle. Triple mutants of Osgsk2, Osgsk3, and Osgsk4, as well as quadruple mutants of all four loci, exhibited reduced plant height and less upright leaves than wild-type siblings. All mutant combinations tested, as well as Osgsk2 single mutations, increased grain size, primarily by increasing grain length. The phenotypes described for the Osgsk CRISPR-Cas9 and Osgsk RNAi lines closely resembled those of maize Zmbin2-RNAi lines, suggesting a conservation of function between maize and rice.

The OsGSK2 protein has also been shown to directly phosphorylate another positive regulator of BR signaling, the GRAS transcription factor encoded by OsDWARF AND LOW-TILLERING (OsDLT) (Tong et al., 2012). Loss-of-function Osdlt mutants have a reduced stature, upright leaves, and reduced tillering (Tong et al., 2009). Overexpression of OsDLT suppressed the phenotypes of OsGSK2 overexpression lines, indicating that OsDLT functions downstream of OsGSK2 in rice (Liu et al., 2021). Another rice GRAS transcription factor, WIDE GRAIN3 (WG3), was shown to physically interact with OsDLT to promote BR signaling and increase grain size (Chen et al., 2022). OsDLT has also been implicated in GA metabolism (Li et al., 2010), but it is unclear whether the effects of Osdlt mutation on GA metabolism are independent of BR signaling or result from crosstalk between these two hormones. The functions of BIN2/GSK3 loci in Setaria and Brachypodium have not been investigated in detail.

BRI1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 (BES1/BZR1)

The BES1/BZR1 genes encode beta-helix-loop-helix (bHLH)-type transcription factors that positively regulate BR signaling (He et al., 2002; Wang et al., 2002; Zhao et al., 2002). There are six BES1/BZR1-like genes in Arabidopsis, 10 in soybean, seven in maize, four in rice, six in Brachypodium, and three in Setaria (Figure 7). AtBES1, AtBZR1, AtBEH1, and AtBEH2 are in the same subclade, along with a single gene from rice and Setaria, two from maize, three from Brachypodium, and six from soybean. AtBEH3 and AtBEH4 belong to a second subclade that contains four soybean genes, two genes from maize, Brachypodium, and rice, and two genes from Setaria. Three maize genes comprise another separate subclade. AtBES1 and AtBZR1 show similar expression profiles across tissues, with AtBES1 most highly expressed in seedling shoots and AtBZR1 most highly expressed in carpels. AtBES1 has the highest average FPKM value across all tissues in Arabidopsis. AtBEH1 is also most highly expressed in seedling shoots, but, interestingly, it has low expression in mature inflorescences, anthers, and carpels. AtBEH2 is also highly expressed in young and mature inflorescences (Figure 7). These data suggest variable gene regulation among the closely related Arabidopsis genes in reproductive structures. AtBEH3 and AtBEH4 also have similar expression profiles across tissues, with the highest expression in young inflorescences. All soybean genes have expression profiles similar to those of their closest Arabidopsis homologs (Figure 7).Figure 7 Phylogeny and transcript abundance across different tissues of the BES1/BZR1 brassinosteroid transcription factor family.

Maximum-approximate-likelihood phylogenetic tree of BES1/BZR1 amino acid sequences from maize (Zm), Setaria (Sevir), rice (Os), Arabidopsis (AT), soybean (Glyma), Brachypodium (Bradi), and the outgroup Physcomitrella (Pp). The relative transcript per million (TPM) values from re-analyzed publicly available datasets of different developmental tissues are represented as a heatmap next to each gene within the family. Yellow indicates the lowest individual transcript abundance in each tissue, and blue indicates the highest abundance in a given tissue. To compare transcript abundance across genes and species within the family, the average FPKM was determined across all tissues analyzed and is presented in the average-FPKM column (left) with the same color distribution described previously. See Supplemental Tables 1–6 for a description of tissue types.

In maize, ZmBES1 and ZmBZR7 are most highly expressed in small, developing tassels. ZmBZR2 has a higher average FPKM value than ZmBES1 and ZmBZR7, which is surprising given its placement in the subclade of AtBEH3 and AtBEH4. ZmBZR1 and ZmBZR7 are also highly expressed in developing maize tassels, but ZmBZR1 is also more highly expressed in young seeds, embryos, and roots relative to the other maize gene expression profiles within subclades with characterized Arabidopsis loci. The three maize genes that comprise their own subclade are most highly expressed in seeds (ZmBZR8) and endosperm (ZmBZR3 and ZmBZR9), suggesting a diversification in the roles of these three BZR genes within maize and compared with other species (Figure 7). However, the very low transcript abundance of these three genes across all tissues could indicate that they are pseudogenes (Figure 7). The four rice BZR transcripts show the highest expression in developing inflorescences and carpels, suggesting their conservation and possible redundancy in rice. The three rice transcripts in the subclade with AtBEH3 and AtBEH4 are also relatively highly expressed in developing embryos compared with other tissues. Brachypodium genes have expression profiles similar to those of their closest homologs in rice and maize (Figure 7).

The first characterized BES1/BZR1 mutant in Arabidopsis, Atbrassinazole-resistant1-1D (Atbzr1-1D), was identified as a dominant allele in a screen for mutants insensitive to BRZ, a BR biosynthesis inhibitor that phenocopies BR-deficient mutants (Asami et al., 2000; He et al., 2002; Wang et al., 2002; Zhao et al., 2002). When grown in the light, the Atbzr1-1D mutants were shorter, had more and wider dark-green leaves, exhibited delayed flowering, and were epistatic to BR biosynthetic and other BR signaling mutants. The AtBZR1, AtBES1, AtBEH1, AtBEH2, AtBEH3, and AtBEH4 proteins all contain a GSK3 phosphorylation site and can be directly phosphorylated by AtBIN2 (Wang et al., 2002; Yin et al., 2002). The GmBEHL1 protein was also shown to interact with GmBIN2 in yeast-two-hybrid and biomolecular fluorescence complementation assays (Yan et al., 2018). Phosphorylation of BES1/BZR1 proteins results in their strict localization to the cytosol (He et al., 2002; Wang et al., 2002; Yin et al., 2002; Zhao et al., 2002). Inhibition or shuttling of phosphorylated BES1/BZR1 proteins out of the nucleus requires their association with 14-3-3 binding proteins (Bai et al., 2007; Gampala et al., 2007; Ryu et al., 2007). Mutation of the AtBIN2 binding site in AtBZR1 abolished 14-3-3 association and increased AtBZR1 localization in the nucleus (Gampala et al., 2007). Studies have also shown that the AtBES1/AtBZR1 transcription factors can be activated independently of the BR signaling pathway (Zheng et al., 2019, 2022; Albertos et al., 2022; Bai et al., 2022; Chen et al., 2022; Shi et al., 2022).

The AtBRZ SENSITIVE SHORT HYPOCOTYL1/AtBLADE ON PETIOLE1 (AtBSS1/AtBOP1) protein was shown to inhibit the nuclear localization of AtBZR1 (Shimada et al., 2015). Overexpression of AtBSS1/AtBOP1 resulted in reduced nuclear localization of AtBZR1, whereas deficient mutants of Atbss1/Atbop1 showed greater AtBZR1 nuclear localization. Dominant and overexpression lines of AtBSS1/AtBOP1 exhibited strong BR-deficient mutant phenotypes (Shimada et al., 2015), further supporting the involvement of AtBSS1/AtBOP1 in inhibition of BR signaling.

Once BRs are present, BIN2 inhibition of BES1/BZR1 transcription factors is removed by subsequent targeting of BIN2 to the 26s proteasome for degradation. Removal of AtBIN2 results in dephosphorylation of AtBES1/AtBZR1 transcription factors by AtPROTEIN PHOSPHATASE2A (AtPP2A) (Di Rubbo et al., 2011; Tang et al., 2011). The unphosphorylated BES1/BZR1 transcription factors can enter the nucleus to bind and regulate BR-responsive genes. The AtBES1/AtBZR1 transcription factors were shown to recruit another bHLH transcription factor, AtBES1 INTERACTING MYC-LIKE1 (AtBIM1), and bind to E-box motifs to promote transcription of BR-responsive genes (Yin et al., 2005). They have also been shown to bind to BR-response elements in the promoters of BR-responsive genes to inhibit transcription (He et al., 2005). The AtBES1/AtBZR1 transcription factors contain an ERF-ASSOCIATED AMPHIPHILIC REPRESSION (EAR) motif near the C-terminal end of the protein that is required for AtBES1/AtBZR1 regulation of gene expression (Wang et al., 2013). Deletion of the EAR motif eliminated their ability to repress gene expression and regulate growth and development, but that activity was recovered by fusing the Groucho/TUP1-LIKE transcriptional corepressor AtTOPLESS (AtTPL) to the EAR motif-less AtBZR1 (Oh et al., 2014). Quadruple mutants of Attpl; Attopless-related1 (Attpr1); Attpr4; Atbzr1-1D suppressed the Atbzr1-1D mutant phenotypes, further indicating the necessity of AtTPL for proper function of AtBZR1 (Oh et al., 2014). Ectopic expression of AtTPL resulted in less upright leaves and altered inflorescence development in an AtBZR1/AtBES1-dependent manner (Espinosa-Ruiz et al., 2017). AtTPL has also been shown to participate in jasmonic acid and auxin signaling (Szemenyei et al., 2008; Pauwels et al., 2010), indicating potential crosstalk between BR signaling and other hormone signaling pathways.

The recessive loss-of-function single mutants Atbzr1, Atbes1, Atbeh3, and Atbeh4 grown in the dark had significantly shorter hypocotyls than their wild-type siblings; however, the reduction was not as severe as that seen in biosynthetic mutants (Lachowiec et al., 2018). In soybean, GmBEHL1 was shown to negatively regulate nodule number but result in larger nodules (Yan et al., 2018). The Atbeh4-1 mutant was the only mutant with a decreased developmental robustness of hypocotyl growth when grown in the dark and was dependent on AtBES1 function (Lachowiec et al., 2018). None of the Atbes1/Atbzr1 mutants influenced developmental robustness when grown in the light. No other obvious vegetative defects were observed in single mutants or in studied double, triple, quadruple, or pentuple mutant combinations of Atbes1/Atbzr1, with the exception of Atbzr1; Atbes1; Atbeh1; Atbeh3; Atbeh4 quintuple mutants, which exhibited curled leaves and a semi-dwarf stature (Lachowiec et al., 2018; Chen et al., 2019). Hextuple mutants of all six Atbes1/Atbzr1 loci exhibited severe dwarf phenotypes and resembled severe Atbri1 recessive mutants. This result indicated a high level of redundancy among all AtBES1/AtBZR1 transcription factors in the regulation of BR-responsive gene expression to control growth and development. Mutant analysis of the GmBZL2 and GmBZL3 genes suggests that this redundancy is conserved between Arabidopsis and soybean (Zhang et al., 2016b; Song et al., 2019b). Notably, AtBEH2 appears to be the weakest of the six AtBES1/AtBZR1 transcription factors, as the pentuple mutant of the other five genes did exhibit a weak phenotype (Chen et al., 2019).

Some mutants of ZmBES1/ZmBZR1 transcription factor genes have been described in maize. UniformMu (Settles et al., 2007) and CRISPR-Cas9 knockout mutants were developed for Zmbes1 and Zmbzr1 (Wang et al., 2022). Both single mutants were significantly shorter and had more erect leaves than wild-type siblings. The Zmbes1 Zmbzr1 double mutant phenotypes were not more severe than that of either single mutant for plant height but were additive for leaf angle, resulting in more erect leaves than those of either single mutant (Wang et al., 2022). This result is surprising, as single mutants of BES1/BZR1 in Arabidopsis do not have a light-grown phenotype, suggesting a diversification of function in maize. Development of higher-order mutant combinations will most likely be necessary to observe a severe phenotype similar to that of biosynthetic mutants.

A recessive mutant of the OsDLT ortholog ZmSCARECROW-LIKE28 (ZmSCL28) was also developed, and the Zmscl28 mutant was shorter and had more erect leaves than either Zmbes1 or Zmbzr1. Double mutants between Zmscl28 and Zmbes1 or Zmbzr1 did not differ in phenotype from Zmscl28, indicating genetic epistasis between ZmSCL28 and ZmBES1/ZmBZR1 (Wang et al., 2022) and conservation between rice and maize.

The ZmBZR2 gene (Yu et al., 2018) was overexpressed in Arabidopsis, where it resulted in larger leaf and seed size (Zhang et al., 2020). The enlargement of these organs was proximal-distal and medial-lateral and was due to larger cell sizes in the overexpression lines. This locus (v3-GRMZM5G852801/GRMZM6G287292; v4-Zm00001d039439) was not included in our phylogenetic and expression analysis (Figure 7) because it was not annotated in version 5 of the maize genome. The ZmBZR6 gene has also been molecularly characterized in maize (Yu et al., 2018). The ZmBZR6 protein contains a bHLH domain and was predicted to bind to BR-response elements and E-box motifs, but it also contains a β-amylase (BAM) domain (Sun et al., 2020). The closest Arabidopsis homologs (AT2G45880 [AtBAM7] and AT5G45300 [AtBAM8]) also contain a BAM domain in addition to the bHLH domain. The Atbam7 Atbam8 double mutants exhibited slower growth and smaller leaves than their wild-type siblings (Reinhold et al., 2011). Mutants of ZmBZR6 exhibited smaller seed size, whereas overexpression of ZmBZR6 in rice resulted in larger seed size (Sun et al., 2021). Overexpression in Arabidopsis reduced ABA sensitivity but enhanced salt and drought tolerance (Sun et al., 2020). These BAM transcription factors were proposed to participate in an alternative signaling pathway other than BR signaling, even though they may compete for the same genetic targets (Reinhold et al., 2011).

The four rice OsBES1/BZR1 genes were characterized using CRISPR-Cas9. Among the single mutants, only Osbzr2 exhibited a phenotype, including shorter plant height, less upright leaves, and suppressed tillering (Liu et al., 2021). Higher-order mutant combinations were obtained by combining weak mutant alleles at one locus with stronger alleles at the others. No double, triple, or quadruple mutants were obtained with OsBZR2, suggesting that these genotypes result in lethality, and this gene encodes the dominant BES1/BZR1 transcription factor in rice. It is perhaps surprising that Osbzr2 was the only single mutant to have a phenotype, as the two maize genes whose single mutants exhibited phenotypes (Wang et al., 2022) were more closely related to the three other rice genes (Figure 7).

The rice U-box E3 ligase OsPUB24 was shown to ubiquitinate OsBZR1, resulting in subsequent degradation of OsBZR1 by the 26S proteasome (Min et al., 2019). Knockout mutants of Ospub24 were hypersensitive to BL treatment, had less upright leaves, and exhibited increased seedling growth of the root and shoot (Min et al., 2019). These results were similar to observations of Atpub39;pub40;pub41 triple mutants in Arabidopsis, which accumulated more AtBZR1 in roots than their wild-type siblings. Consistent with this observation, overexpression of OsPUB40 resulted in lower AtBZR1 levels in a root-specific manner (Kim et al., 2019). No mutants of BES1/BZR1 have been described in Brachypodium or Setaria, and, given the conserved redundancy of gene function described in Arabidopsis, soybean, and rice, higher-order mutant combinations will most likely be necessary to identify their functions.

Supporting citations

The following references appear in the supplemental information: Babiychuk et al. 2008; Bolser et al., 2016; Cheng et al. 2021; Davidson et al. 2012; Deng et al. 2017; Go et al. 2012; Goodstein et al. 2012; Harrop et al. 2019; Hu et al. 2012; Huson and Scornavacca 2012; Kim et al. 2013; Kim et al. 2023; Kim et al. 2011; Kolesnikova et al. 2006; Kovaka et al. 2019; Krishnakumar et al. 2017; Langmead and Salzberg 2012; Larkin et al. 2007; Li et al. 2009b; Li et al. 2016; Liu et al. 2021b; Liu et al. 2015; Maddison and Maddison 2019; Maselli et al. 2014; Mergner et al. 2020; Mora-Garcia et al. 2004; Muto et al. 2004; Novikova et al. 2021; Parvathaneni et al. 2020; Pollier et al. 2019; Portwood et al. 2019; Posé et al. 2009; Price et al. 2010; Qi et al. 2012; Ryu et al. 2010; Severin et al. 2010; Stelpflug et al. 2016; Tang et al. 2008; Tian et al. 2023; Wang et al. 2017; Wang et al. 2011; Yin et al. 2022; Zhang et al. 2012b; Zhou and Li 2005; Zhu et al. 2018.

Funding

Mention of trade names or commercial products in this publication was solely for the purpose of providing specific information and does not imply recommendation or endorsement by the 10.13039/100000199 US Department of Agriculture . The US Department of Agriculture is an equal opportunity provider and employer. This research used resources provided by the SCINet project of the 10.13039/100007917 USDA Agricultural Research Service , ARS project number 0500-00093-001-00-D . This work was supported by 10.13039/100000199 USDA Hatch project number IOW03649 (E.V.) and by the Department of Agriculture, 10.13039/100005825 National Institute of Food and Agriculture (USDA-NIFA) fellowship #2019-67012-29655 (N.B.B.).

Author contributions

B.Z., E.V., and N.B.B. wrote the manuscript.

Supplemental information

Document S1. Supplemental Figures 1–5, Supplemental Tables 1–6, and materials and methods

Document S2. Article plus supplemental information

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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