Reciprocal inhibition of expression between RAV1 and BES1 modulates plant growth and development in Arabidopsis.

RAV1 (Related to ABI3/VP1) is a plant-specific B3 and AP2 domain-containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large-scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1-EMS-SUPPRESOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1), which are critical transcription factors for the BR-signaling process. Using RAV1-overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1-D were attenuated by the repression of endogenous BES1 expression in transgenic bes1-D plants overexpressing RAV1. RNA-sequencing analysis of RAV1-overexpressing transgenic plants and bes1-D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co-regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co-regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 Modulates Abscisic Acid Signaling by Inducing PYR1 Monomerization and Association With ABI1 in Arabidopsis.

Brassinosteroid-Insensitive 1-Associated Receptor Kinase 1 (BAK1) is a versatile kinase involved in many different plant developmental responses. Previously, we showed that BAK1 interacts with open stomata 1 (OST1), a cytoplasmic kinase, to promote abscisic acid (ABA)-induced stomatal closure. ABA is a plant hormone that primarily regulates stress responses and is recognized by the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENT OF ABA RECEPTORS (RCAR), which activates ABA signaling. Here, we demonstrated that BAK1 interacts with PYR1 and phosphorylates PYR1 in response to ABA in plants. We identified T137 and S142 of PYR1 as the phosphosites targeted by BAK1. Using phosphomimetic (PYR1DD) and phospho-dead (PYR1AA) PYR1 compared with wild-type PYR1, we showed that transgenic plants overexpressing a phosphomimetic PYR1 exhibited hypersensitivity to the inhibition of ABA-induced root growth and seed germination and increased ABA-induced stomatal closure and ABA-inducible gene expression. As underlying reasons for these phenomena, we further demonstrated that phosphorylated PYR1 existed in a monomeric form, in which ABA binding was increased, and the degree of complex formation with ABI1 was also increased. These results suggest that BAK1 positively modulates ABA signaling through interaction with PYR1, in addition to OST1.

BAK1-induced RPK1 phosphorylation is essential for RPK1-mediated cell death in Arabidopsis.

Being sessile, plants must deploy highly exquisite systems to respond to various internal and external signals for modulating growth and development throughout their lifespan. Many studies on Arabidopsis have shown that leucine-rich repeat-containing receptor-like kinases, including BRI1-associated receptor kinase 1 (BAK1) and receptor-like protein kinase 1 (RPK1), are suitable for such pleiotropic demands of plants. Previously, BAK1 and RPK1 were independently proven to be involved in the regulation of premature cell death. BAK1 inhibits spontaneous cell death and promotes defense-induced cell death. Meanwhile, RPK1 mediates reactive oxygen species (ROS) production through complexation with CaM4 and RbohF in an age-dependent manner. In the present study, RPK1-induced cell death and growth retardation were abolished both with respect to the phenotype and ROS production in bak1 mutants. Moreover, BAK1 interacts with RPK1 and mediates its unidirectional phosphorylation in plants. Further, BAK1-mediated RPK1 phosphorylation is indispensable for RPK1-CaM4 interaction, which is vital for ROS production, resulting in cell death. The presence of BAK1 enhanced the expression of cell death- and senescence-related genes, such as ORE1, PR1, SAG12, and SIRK in RPK1-mediated signaling cascades. Overall, in Arabidopsis, in addition to independent cell death regulation by BAK1 and RPK1, multiple-layers control cell death and premature senescence via the coordinated action of BAK1 and RPK1.

Open stomata 1 exhibits dual serine/threonine and tyrosine kinase activity in regulating abscisic acid signaling.

Open Stomata 1 (OST1)/SnRK2.6 is a critical component connecting abscisic acid (ABA) receptor complexes and downstream components, including anion channels and transcription factors. Because OST1 is a serine/threonine kinase, several autophosphorylation sites have been identified, and S175 is known to be critical for its kinase activity. We previously reported that BAK1 interacts with and phosphorylates OST1 to regulate ABA signaling. Here, we mapped additional phosphosites of OST1 generated by autophosphorylation and BAK1-mediated transphosphorylation in Arabidopsis. Many phosphosites serve as both auto- and transphosphorylation sites, especially those clustered in the activation loop region. Phospho-mimetic transgenic plants containing quadruple changes in Y163, S164, S166, and S167 rescued ost1 mutant phenotypes, activating ABA signaling outputs. Moreover, we found that OST1 is an active tyrosine kinase, autophosphorylating the Y182 site. ABA induced tyrosine phosphorylation of Y182 in OST1; this event is catalytically important for OST1 activity in plants. ABA-Insensitive 1 (ABI1) and its homologs ABI2 and HAB1, PP2C serine/threonine phosphatases that are known to dephosphorylate OST1 at S175, function as tyrosine phosphatases acting on the phosphorylated Y182 site. Our results indicate that phosphorylation cycles between OST1 and ABI1, which have dual specificity for tyrosine and serine/threonine, coordinately control ABA signaling in Arabidopsis.

Receptor-like protein kinases RPK1 and BAK1 sequentially form complexes with the cytoplasmic kinase OST1 to regulate ABA-induced stomatal closure.

Regulation of plant water status occurs via abscisic acid (ABA)-induced stomatal closure. Open Stomata 1 (OST1) is a critical ABA signaling component regulating this process in guard cells. We previously reported that BRI1-associated receptor kinase 1 (BAK1) positively regulates ABA-induced stomatal closure by interacting with and phosphorylating OST1. Here, using Arabidopsis, we show that the receptor-like protein kinase 1 (RPK1), previously known to be induced by ABA, is a positive ABA-signaling component in guard cell movement, and interacts with OST1. ABA-inducible expression patterns were observed in RPK1 and OST1, but not in BAK1. We investigated the underlying mechanisms by which the RPK1-OST1 and BAK1-OST1 complexes interact in stomatal guard cells by monitoring the complex formation continuously using fluorescence resonance energy transfer analyses. We found that the BAK1-OST1 complex was formed earlier than the RPK1-OST1 complex in response to ABA. In vitro and semi-in vivo kinase assays revealed that a transphosphorylation event occurred in the RPK1-OST1 complex, which differs from that in the BAK1-OST1 complex, wherein only OST1 phosphorylation occurred via BAK1. ABA-insensitive 1 (ABI1) only dephosphorylated OST1 in the BAK1-OST1 complex, but dephosphorylated both RPK1 and OST1 proteins in the RPK1-OST1 complex. Our results suggest that there are multiple coordinated ABA signaling systems to regulate stomatal movement.

Brassinosteroid reduces ABA accumulation leading to the inhibition of ABA-induced stomatal closure.

Proper regulation of stomatal movement in response to various environmental stresses or developmental status is critical for the adaptation of many plant species to land. In plants, abscisic acid (ABA)-induced stomatal closure is a well-adapted method of regulating water status. In addition to ABA, we previously showed that plant-specific steroidal hormone, brassinosteroid (BR), also induces stomatal closure; however, BR modulates ABA-induced stomatal closure negatively at high concentrations. In this study, we further investigated the cross-talk between ABA and BR in relation to stomatal movement. In contrast to previous reports that ABA-induced stomatal closure was inhibited by brassinolide (BL), the most active BR, we showed that BL-induced stomatal closure was enhanced by ABA, indicating that the sequence of ABA or BL treatments led to different results. We found that this phenomenon occurred because the guard cells still had the capacity to be closed further by ABA, as the degree of stomatal closure by BL was always less than that by ABA. We also found that BL-induced stomatal closure required Open Stomata 1 (OST1) activity and the induced expression of OST1 was indifferent to the sequence of ABA and/or BL treatments. In addition, we examined the underlying mechanism by which inhibition of ABA-induced stomatal closure by BL occurred. We revealed that the downregulation of ABA-biosynthetic genes by BL resulted in a lower accumulation of ABA. These results suggested that the regulation of stomatal movement is finely controlled by the combined effects of plant hormones, ABA and BR.