Leaf size strongly influences the efficiency of photosynthesis in plants (Gonzalez et al. 2012). Brassinosteroids (BRs) are essential phytohormones that promote plant growth and a characteristic phenotype of BR-related mutants is reduced leaf size (Mao and Li 2020; Li and Chory 1997; Li et al. 1996; Praveena et al. 2020; Yang et al. 2011; Clouse and Sasse 1998; Nolan et al. 2017; Vert et al. 2005). In BR biosynthesis mutant constitutive photomorphogenesis and dwarfism (cpd), the leaves are small, round, and dark-green (Noguchi et al. 1999; Szekeres et al. 1996). Another BR-deficient mutant, deetiolated-2 (det2), has leaves that are small and round and twice as numerous as the wild type (Azpiroz et al. 1998; Chory et al. 1991). The transmembrane receptor kinase brassinosteroid insensitive1 (BRI1), has been associated with BR responses (Guo et al. 2013). Mutations in BRI1 result in BR-insensitivity and a morphological phenotype almost identical to that of the BR biosynthesis mutant cpd (Clouse et al. 1996). In rice, M107 is a gain-of-function mutant of the BR biosynthetic gene P450 CYP724B1 with a typical BR excess phenotype, including long and narrow leaves with greatly increased leaf angles (Wan et al. 2009). In addition, the positive regulatory protein complexes of the BR signaling pathway reduced leaf angle 1 (RLA1) and BRASSINAZOLE RESISTANT1 (OsBZR1) have mutants with a distinct, erect leaf phenotype (Qiao et al. 2017). In Gossypium hirsutum, GhPAG1 is highly homologous to AtCYP734A1, and its expression is activated in pagoda1 (pag1) mutants exhibiting dwarfism and smaller leaf size due to inhibition of cell expansion (Yang et al. 2014).
BR regulates expression of downstream genes mainly through the transcription factor (TF) BES1/BZR1 (BRI1-EMS-SUPPRESSOR 1/BRASSINAZOLE-RESISTANT 1) interacting with key elements, including E-box motif (CANNTG) and BR response element (BRRE; CGTGC/TG) (Li et al. 2018). BES1/BZR1 can directly bind to the promoter regions of CPD, dwarf4 (DWF4), and BR-6-oxidase (BR6OX) to regulate cell expansion in leaves (He et al. 2005). The bes1-D mutant, whose BES1 protein is widely expressed by an amino acid mutation, exhibits a constitutive BR response phenotype including long, bending petioles and curled leaves. Notably, multiple BR-induced genes are up-regulated or hyperresponsive to BR in bes1-D, including EXORDIUM (EXO), which is described as an AtPhi-1 phosphate-induced protein (Yin et al. 2002). BZR1/BES1 genes have been identified from many species such as Chinese cabbage (Wu et al. 2016), maize (Yu et al. 2018b), rice (Bai et al. 2007), and cotton (Liu et al. 2018) and they are reported to have a conserved role in regulating BR signal transduction pathway.
The EXO gene was first reported as a plant growth regulator expressed in tissues with high auxin concentrations (Farrar et al, 2003). In addition, as a BR response gene, EXO expression is induced after exogenous 2,4-epibrassinolide treatment in Arabidopsis (Schröder et al. 2009; Coll-Garcia et al. 2004). Proteomics approaches identified the EXO, EXL1, EXL2, EXL3, and EXL4 proteins as part of the cell wall proteome and determined that they promote plant growth, cell expansion, and carbon starvation response through mediating BR (Schröder et al. 2012; Schröder et al. 2009; Coll-Garcia et al. 2004). In Arabidopsis, rosette leaves became larger in EXO overexpression transgenic plants compared with wild type, and increased transcript levels of BR response genes were observed (Coll-Garcia et al. 2004). However, elongation of abaxial epidermal and palisade cells in the subepidermal layer were inhibited in the exo knockout mutant based on scanning electron microscopy (SEM) showing a diminished leaf and root growth phenotype and a diminished response to BL (Schröder et al. 2009). OsEXO knockout plants exhibited partial dwarfism and had smaller cells in the culms, promoting cell expansion by regulating microtubule organization (Aya et al. 2014). EgEXO overexpression in Eucalyptus globulus promoted plant height and increased leaf biomass. In addition, the presence of two G-boxes in the promoter region suggests that it is regulated by the BES1 family of TFs (Sousa et al. 2020).
Cotton (mainly upland cotton, G. hirsutum) is a primary fiber crop and an excellent model to study polyploidization, genome evolution, and cell expansion (Malik et al. 2018; Ali et al. 2021). Cotton leaf size is an important agronomic trait that affects plant architecture, yield, and stress tolerance (Andres et al. 2016). Cotton leaf size has significant phenotypic diversity in crops and has a unique role in cotton development (Dolan and Poethig 1991). Only a few genes affecting cotton leaf development have been functionally characterized, such as LMI1 (LATE MERISTEM IDENTITY1, an HD-Zip transcription factor) and GhARF16 (Auxin response factor) (He et al. 2021; Andres et al. 2017). However, the mechanisms of cotton leaf development remain largely unknown.
Previous studies have shown that BR primarily regulates leaf size by affecting cell expansion, but the genetic mechanisms involved are unclear. In this study, the major TF GhBES1 in the BR signaling pathway was shown to act as a key regulator that binds to the GhEXO2 promoter and increases its expression, enhancing cell expansion and ultimately affecting leaf size. In brief, our data suggest that GhEXO2, the downstream target gene of GhBES1, functions in expanding cotton leaf cells in BR signaling to regulate leaf size.