Professor Baowen Zhang and Professor Wenqiang Tang from the Key Laboratory of Molecular and Cellular Biology of the Ministry of Education at Hebei Normal University published a research article entitled “BSK family kinases are essential for brassinosteroid signaling and suppression of adventitious rooting by repressing the expression of LBD16” in the internationally renowned plant science journal New Phytologist.
During the study, plants were cultivated in a Jiupo Artificial Climate Chamber under controlled environmental conditions, including a temperature of 22°C, relative humidity of approximately 55%, a 16 h light / 8 h dark photoperiod (long-day conditions), and a light intensity maintained at 100–120 μmol·m⁻²·s⁻¹.
The study generated an Arabidopsis BSK-family undecuple mutant, revealing that BSK family members exhibit a high degree of functional redundancy yet are indispensable for brassinosteroid (BR) signaling. Furthermore, the researchers uncovered a novel regulatory mechanism whereby BR signaling suppresses adventitious root (AR) formation through the BSK–BZR1 signaling module, which represses the expression of LBD16.
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To investigate the functions of the twelve members of the BSK family, the research team first examined the promoter activities of eleven BSK genes (excluding BSK7) using a GUS reporter system. The results showed that BSK12 was specifically expressed in sperm cells, whereas the remaining family members exhibited distinct expression patterns across seedlings, roots, leaves, inflorescences, and anthers (Figure 1).
Subsequently, individual BSK genes were overexpressed in the weak BR-insensitive mutant bri1-5. Most BSK members partially rescued the dwarf phenotype to varying degrees, with BSK3 and BSK4 displaying the strongest effects (Figure 2). These findings indicate that BSK family members are highly functionally redundant and that most act as positive regulators of BR signaling.
Because of the extensive functional redundancy within the BSK family, mutations in single genes or a small number of genes are insufficient to produce obvious BR-deficient phenotypes. Through multiple rounds of genetic crossing combined with CRISPR/Cas9-mediated genome editing, the researchers progressively generated octuple, nonuple, and ultimately the undecuple mutant bsk-u. In this mutant, the functions of BSK1–8 and BSK10–12 were completely abolished or substantially reduced, while BSK9 expression was markedly downregulated.
Phenotypic analyses revealed that bsk-u plants exhibited severe BR-insensitive characteristics, including extreme dwarfism, dark-green curled leaves, and shortened petioles, closely resembling the phenotype of the BR receptor-null mutant bri1-116. Molecular analyses further demonstrated that bsk-u plants were completely unresponsive to BR treatment, with the key transcription factor BZR1 remaining constitutively hyperphosphorylated and inactive (Figure 3). Collectively, these results provide compelling genetic evidence that the BSK family constitutes an essential core component of BR signal transduction.
To determine the position of BSKs within the signaling pathway, the researchers introduced either BSK3 or the constitutively active transcription factor bzr1-1D into the bsk-u mutant background. Overexpression of BSK3 fully restored the mutant phenotype to a wild-type state (Figure 4), whereas expression of bzr1-1D partially alleviated dwarfism and growth defects (Figure 5). These findings not only confirm that the bsk-u phenotype results from the loss of BSK function but also demonstrate that BSK kinases function upstream of BZR proteins and regulate plant growth through activation of BZR1.
Transcriptome sequencing (RNA-seq) revealed widespread disruption of BR-responsive gene expression in the bsk-u mutant, including numerous genes associated with root development. Gene Ontology (GO) enrichment analysis further suggested a close relationship between BR signaling and adventitious root development.
Using a leaf explant-based adventitious root induction system, the researchers found that:
• Wild-type (Col-0): Exogenous BR treatment strongly inhibited adventitious root formation, whereas treatment with the BR biosynthesis inhibitor propiconazole (PCZ) promoted adventitious rooting.
• BR signaling-defective mutants: Both bsk-u and bri1-5 produced significantly more adventitious roots than wild-type plants and were insensitive to BR treatment.
• BR signaling-activated mutant: The constitutively activated BR signaling mutant bin2 completely failed to generate adventitious roots (Figure 7).
These genetic and physiological analyses provide strong evidence that the BR signaling pathway functions as a negative regulator of adventitious root formation.
To elucidate the underlying molecular mechanism, the research team focused on LBD16, a key regulator of adventitious root development. Expression analyses demonstrated that BR significantly suppressed LBD16 expression in wild-type plants; however, this repression was abolished in the bsk-u mutant.
Dual-luciferase assays further showed that the core BR signaling transcription factor BZR1 directly binds to the LBD16 promoter and represses its transcriptional activity. Genetic analyses revealed that LBD16 loss-of-function mutants were unable to produce adventitious roots when BR biosynthesis was inhibited, whereas LBD16 overexpression partially restored adventitious root formation suppressed by BR treatment (Figure 8).
Taken together, this study establishes a complete signaling cascade in which:
BR → BRI1 → BSKs → BZRs ⟶ repression of LBD16 ⟶ regulation of adventitious root formation (Figure 9).
These findings not only demonstrate the indispensable role of the BSK family in BR signal transduction but also uncover a previously unrecognized molecular mechanism by which BR signaling suppresses adventitious rooting through direct repression of LBD16 expression.
