Hemostatic Effect and Mechanism of Epidermal Growth Factor-Hemostatic Powder after Endoscopic Submucosal Dissection in a Porcine Model.

Background/Aims: Among several methods used to prevent endoscopic submucosal dissection (ESD) bleeding, the recently developed hemostatic powder (HP) has few technical limitations and is relatively easy-to-use. This study aimed to analyze the hemostatic effects and mechanisms of two HPs using a porcine upper gastrointestinal hemorrhage model. Methods: We evaluated HPs (Endospray and epidermal growth factor [EGF]-endospray) for adhesion, waterproofing ability, permeability, and absorption in vitro. ESD was performed to induce bleeding ulcers in the porcine stomachs. In a total of three pigs, three bleeding ulcers per animal were generated. Hemostasis and rebleeding were evaluated endoscopically. After 72 hours, the animals were sacrificed, and histologically analyzed. Results: The water absorption of HPs was over 20 times the initial value within 30 minutes. The gelated HPs completely blocked water penetration into the applied site within 5 minutes and strongly adhered to the Petri-dish surface for up to 6 hours. The initial hemostasis rates within 5 minutes were 33.3%, 100.0%, and 66.7%, and the rebleeding rates at 6 to 72 hours after HP application were 33.3%, 16.7%, and 33.3% (control, Endospray, and EGF-endospray groups, respectively). Histological analysis revealed the thickness of the regenerated mucosa (522.1, 514.5, and 680.3 µm) and the submucosal layer (1,510.3, 2,848.2, and 3,062.3 µm) and the number of newly formed blood vessels (15.3, 17.9, and 20.5) in the control, Endospray, and EGF-endospray groups, respectively. Conclusions: The endoscopic HPs demonstrated the ability to elicit effective initial hemostasis and the histological ulcer-healing effect of EGF in an animal model of hemorrhagic gastric ulcers.

Safety and Efficacy of Recombinant Human Bone Morphogenetic Protein-2 in Multilevel Posterolateral Lumbar Fusion in a Prospective, Randomized, Controlled Trial.

OBJECTIVE: This study is an investigator-initiated, prospective, randomized, controlled study to evaluate the efficacy and safety of the combined use of recombinant human BMP-2 (rhBMP-2) and a hydroxyapatite (HA) carrier in multilevel fusion in patients with adult spinal deformity (ASD). METHODS: Thirty patients underwent posterolateral fusion for lumbar spinal deformities at 3 to 5 segments between L1 and S1. The patients received rhBMP-2+HA or HA on the left or right side of the transverse processes. They were followed up regularly at 1, 3, 6, and 12 months postoperatively. Fusion was defined according to the bone bridging on computed tomography scans. The fusion rate per segment was subanalyzed. Function and quality of life as well as pain in the lower back and lower extremities were evaluated. RESULTS: The union rate for the rhBMP-2+HA group was 100% at 6 and 12 months. The union rate for the HA group was 77.8% (21 of 27) at 6 months and 88.0% (22 of 25) at 12 months (p = 0.014 at 6 months; not significant at 12 months). All segments were fused at 6 and 12 months in the rhBMP-2+HA group (p < 0.001). In the HA group, 108 of 115 segments (93.5%) were fused at 6 months and 105 of 109 segments (96.3%) at 12 months. Other clinical parameters (visual analogue scale, 36-item Short Form Health Survey, and Scoliosis Research Society-22 scores) improved compared to baseline. CONCLUSION: Combining rhBMP-2 and an HA carrier is a safe and effective method to achieve multilevel fusion in patients with ASD.

Performance evaluation of biodegradable polymer sirolimus and ascorbic acid eluting stent systems.

The purpose of this study was to evaluate the performance of biodegradable polymer sirolimus and ascorbic acid eluting stent systems with four commercially available drug-eluting stents (DES). We investigated the characterization of mechanical properties by dimension, foreshortening, recoil, radial force, crossing profile, folding shape, trackability, and dislodgement force. Additionally, we identify the safety and efficacy evaluation through registry experiments. Each foreshortening and recoil of D + Storm® DES is 1.3 and 3.70%, which has better performance than other products. A post-marketing clinical study to evaluate the performance and safety of D + Storm® DES is ongoing in real-world clinical settings. Two hundred one patients were enrolled in this study and have now completed follow-up for up to 1 month. No major adverse cardiovascular event (MACE) occurred in any subjects, confirming the safety of D + Storm® DES in the clinical setting. An additional approximately 100 subjects will be enrolled in the study and the final safety profile will be assessed in 300 patients. In conclusion, this study reported the objective evaluation of DES performance and compared the mechanical responses of four types of DES available in the market. There is little difference between the four cardiovascular stents in terms of mechanical features, and it can help choose the most suitable stent in a specific clinical situation if those features are understood. Graphical abstract.

Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases.

Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development and immunity in plants, but the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLAGELLIN SENSING 2 (FLS2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We surprisingly found that the loss of the BSU1 family phosphatases activates effector-triggered immunity and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine 251. Mutation of serine 251 reduces BSU1's ability to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress effector-triggered immunity and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine 764 upon brassinosteroid signalling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signalling specificities for diverse plant receptor kinases.

Brassinosteroids enhance salicylic acid-mediated immune responses by inhibiting BIN2 phosphorylation of clade I TGA transcription factors in Arabidopsis.

Salicylic acid (SA) plays an important role in plant immune response, including resistance to pathogens and systemic acquired resistance. Two major components, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPRs) and TGACG motif-binding transcription factors (TGAs), are known to mediate SA signaling, which might also be orchestrated by other hormonal and environmental changes. Nevertheless, the molecular and functional interactions between SA signaling components and other cellular signaling pathways remain poorly understood. Here we showed that the steroid plant hormone brassinosteroid (BR) promotes SA responses by inactivating BR-INSENSITIVE 2 (BIN2), which inhibits the redox-sensitive clade I TGAs in Arabidopsis. We found that both BR and the BIN2 inhibitor bikinin synergistically increase SA-mediated physiological responses, such as resistance to Pst DC3000. Our genetic and biochemical analyses indicated that BIN2 functionally interacts with TGA1 and TGA4, but not with other TGAs. We further demonstrated that BIN2 phosphorylates Ser-202 of TGA4, resulting in the suppression of the redox-dependent interaction between TGA4 and NPR1 as well as destabilization of TGA4. Consistently, transgenic Arabidopsis overexpressing TGA4-YFP with a S202A mutation displayed enhanced SA responses compared to the wild-type TGA4-YFP plants. Taken together, these results suggest a novel crosstalk mechanism by which BR signaling coordinates the SA responses mediated by redox-sensitive clade I TGAs.

Irradiation with 590-nm yellow light-emitting diode light attenuates oxidative stress and modulates UVB-induced change of dermal fibroblasts.

Recently, light-emitting diode (LED)-based devices have emerged as effective and safe tools for the treatment of photoaged skin. However, few studies have been conducted to elucidate the underlying mechanism behind the effect on photoageing of LED light. In this study, we induced photoageing of human dermal fibroblasts (HDFs) with Ultraviolet B (UVB) irradiation and evaluated the ability of 590-nm LED radiation to induce recovery from oxidative stress, restore collagen formation and regulate inflammatory changes. Photoageing was induced in cultured human dermal fibroblasts (HDFs) using UVB irradiation of 50 mJ/cm2 . Then, the photoaged HDFs were irradiated with LED using a custom-built 590-nm LED device which emits light with an intensity of 38 mW/cm2 (irradiated for 900 s with 34.2 J/cm2 of total energy). LED irradiation significantly attenuated UVB-induced reactive oxygen species generation and UVB-induced phosphorylation of JNK, c-Fos and c-Jun. In addition, the procollagen levels were recovered significantly, and MMP-9 levels were significantly suppressed after LED irradiation. The UVB-induced phosphorylation levels of NF-κB and pro-inflammatory enzyme COX-2 also significantly decreased. Our results suggest that 590-nm yellow light irradiation may be an effective and safe anti-oxidative and anti-inflammatory treatment modality for photoaged skin.

A First-in-Man Clinical Evaluation of Sirolimus and Ascorbic Acid-Eluting Stent Systems: a Multicenter, Subject-Blinded, Randomized Study.

BACKGROUND AND OBJECTIVES: This clinical trial was conducted to evaluate the safety and efficacy of D+Storm™ drug-eluting stent (DES) and BioMatrix Flex™ DES. METHODS: This study was a multicenter, subject-single-blind, randomized, and confirmed comparative clinical trial. According to the inclusion criteria, those diagnosed with stable angina, unstable angina, silent ischemia, or non-ST-segment myocardial infarction were selected among patients with coronary artery stenosis as subjects. Among the subjects with 50% stenosis on coronary angiography, the experiment was performed on those who had a lesion with reference vessel 2.5-4.0 mm in diameter and ≤40 mm in length. The primary endpoint was an in-segment late loss and the secondary endpoints were in-stent late lumen loss, stent malapposition, the incidence of mortality, myocardial infarction, reoperation, and stent thrombosis at 36 weeks. RESULTS: 57 patients in the D+Storm™ DES group and 55 patients in the BioMatrix Flex™ DES group were enrolled in the study. Fifty-seven patients in the D+Storm™ DES group and Fifty-five patients in the BioMatrix Flex™ DES group were enrolled in the study. An average of in-segment late lumen loss was 0.08±0.13 mm in the D+Storm™ DES group and 0.14±0.32 mm in the BioMatrix Flex™ DES group with no significant difference between the 2 groups (p=0.879). In addition, there was no significant difference in adverse events between D+Storm™ DES and BioMatrix Flex™ DES. CONCLUSIONS: This study demonstrated the clinical effectiveness and safety of D+Storm™ DES implantation in patients with coronary artery disease over a 36-week follow-up period.

Endogenous level of abscisic acid down-regulated by brassinosteroids signaling via BZR1 to control the growth of Arabidopsis thaliana.

The increased level of endogenous abscisic acid (ABA) in brassinosteroid (BR)-deficient mutants, such as det2 and cyp85a1 × cyp85a2, suggests that ABA synthesis is inhibited by endogenous BRs in Arabidopsis thaliana. Expression of the ABA biosynthesis gene ABA-deficient 2 (ABA2) was negatively regulated by exogenously applied BR but up-regulated by the application of brassinazole and in det2 and cyp85a1 × cyp85a2. In addition, ABA2 expression decreased in bzr1-1D, showing that ABA biosynthesis is inhibited by BR signaling via BZR1, intermediated by ABA2, in Arabidopsis. Four cis-element sequences (E-boxes 1-4) in the putative promoter region of ABA2 were identified as BZR1 binding sites. The electrophoretic mobility shift assay and chromatin immune precipitation analysis demonstrated that BZR1 directly binds to overlapped E-boxes (E-box 3/4) in the promoter region of ABA2. The level of endogenous ABA was decreased in bzr1-1D compared to wild-type, indicating that binding of BZR1 to the ABA2 promoter inhibits ABA synthesis in Arabidopsis. Compared to wild-type, aba2-1 exhibited severely reduced growth and development. The abnormalities in aba2-1 were rescued by the application of ABA, suggesting that ABA2 expression and ABA synthesis are necessary for the normal growth and development of A. thaliana. Finally, bzr1-KO × aba2-1 exhibited inhibitory growth of primary roots compared to bzr1-KO, verifying that ABA2 is a downstream target of BZR1 in the plant. Taken together, the level of endogenous ABA is down-regulated by BR signaling via BZR1, controlling the growth of A. thaliana.

Brassinosteroids signaling via BZR1 down-regulates expression of ACC oxidase 4 to control growth of Arabidopsis thaliana seedlings.

ProACO4-GUS expression and RT-PCR analysis revealed that ACO4 is predominantly expressed in shoots of Arabidopsis seedlings under light conditions. ACO4-overexpressed mutant 35S-ACO4 produced more ethylene relative to the wild-type, which resulted in reduced growth of Arabidopsis seedlings. The abnormal growth of seedlings recurred after the application of Co2+ ions, suggesting that ACO4 is a functional ACO necessary to regulate the growth and development of Arabidopsis seedlings. Exogenously-applied brassinosteroids (BRs) inhibited the expression of ACO4, and an enhanced ACO4 expression was found in det2, a BR-deficient mutant. Additionally, expression of ACO4 was decreased in bzr1-D (a BZR1-dominant mutant), implying that BR signaling negatively regulates ACO4 expression via BZR1 in Arabidopsis. In the intergenic region of ACO4, four E-boxes and a BR regulatory element (BRRE) are found. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that BZR1 binds directly to the BRRE in the putative promoter region of ACO4. By binding of BZR1 to BRRE, less ethylene was produced, which seems to regulate the growth and development of Arabidopsis seedlings.

Establishment of Biosynthetic Pathways To Generate Castasterone as the Biologically Active Brassinosteroid in Brachypodium distachyon.

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that castasterone and its biosynthetic precursors are found in Brachypodium distachyon. In vitro conversion experiments with crude enzyme solutions prepared from B. distachyon demonstrated the presence of the following biosynthetic sequences: campesterol → campesta-4-en-3-one → campesta-3-one → campestanol → 6-deoxocathasterone → 6-deoxoteasterone → teasterone ↔ 3-dehydroteasterone ↔ typhasterol → castasterone. campesterol → 22-hydroxycampesterol → 22-hydroxy-campesta-4-en-3-one → 22-hydroxy-campesta-3-one → 6-deoxo-3-dehydroteasterone → 3-dehydroteasterone. 6-deoxoteasterone ↔ 6-deoxo-3-dehydroteasterone ↔ 6-deoxotyphasterol → 6-deoxocastasterone → castasterone. This shows that there are campestanol-dependent and campestanol-independent pathway in B. distachyon that synthesize 24-methylated brassinosteroids (BRs). Biochemical analysis of BRs biosynthetic enzymes confirmed that BdDET2, BdCYP90B1, BdCYP90A1, BdCYP90D2, and BdCYP85A1 are orthologous to BR 5α-reductase, BR C-22 hydroxylase, BR C-3 oxidase, BR C-23 hydroxylase, and BR C-6 oxidase, respectively. Brassinolide was not identified in B. distachyon. Additionally, B. distachyon crude enzyme solutions could not catalyze the conversion of castasterone to brassinolide, and the gene encoding an ortholog of CYP85A2 (a brassinolide synthase) was not found in B. distachyon. These results strongly suggest that the end product for brassinosteroid biosynthesis which controls the growth and development of B. distachyon is not brassinolide but rather castasterone.

In Vitro Analysis of Histology, Mechanics, and Safety of Radiation-free Pre-hydrated Human Acellular Dermal Matrix.

PURPOSE: Acellular dermal matrix (ADM) supports tissue expanders or implants in implant-based breast reconstruction. The characteristics of ADM tissue are defined by the manufacturing procedure, such as decellularization, preservation, and sterilization, and are directly related to clinical outcomes. This study aimed to compare the properties of a new pre-hydrated-ADM (H-ADM-low) obtained using a decellularization reagent reduction process with a low concentration of detergent with those of radiation-sterilized H-ADM and freeze-dried ADM (FD-ADM). METHODS: ADMs were evaluated in terms of structure, mechanical quality, and cytotoxicity using histochemical staining, tensile strength testing, and in vitro cell viability analysis. RESULTS: The tissue structure of H-ADM-low (CGDERM ONE-STEP) was similar to that of native skin despite complete decellularization. By contrast, in FD-ADM, the tissue structure was damaged by the freeze-drying process, and radiation-sterilized H-ADM showed a compact fibrillar arrangement. Furthermore, matrix components such as collagen and elastin were preserved in H-ADM-low, whereas a loss of elastin fibers with fragmented distribution was observed in radiation-sterilized H-ADMs. H-ADM-low's tensile strength (58.84 MPa) was significantly greater than that of FD-ADM (38.60 MPa) and comparable with that of radiation-sterilized H-ADMs. The residual detergent content in H-ADM-low (47.45 mg/L) was 2.67-fold lower than that of H-ADM decellularized with a conventional detergent concentration (126.99 mg/mL), and this finding was consistent with the cell viability results (90.7% and 70.7%, respectively), indicating that H-ADM-low has very low cytotoxicity. CONCLUSIONS: H-ADM-low produced through aseptic processes retains the original tissue structure, demonstrates excellent mechanical properties, and does not affect cell viability. Therefore, this newer H-ADM is suitable for use in implant-based breast reconstruction.

BES1 directly binds to the promoter of the ACC oxidase 1 gene to regulate gravitropic response in the roots of Arabidopsis thaliana.

Brassinosteroids (BRs) are known to be endogenous regulators of ethylene production, suggesting that some BR activity in plant growth and development is associated with ethylene. Here, we demonstrated that ethylene production in Arabidopsis thaliana roots is increased by BR signaling via the ethylene biosynthetic gene for ACC oxidase 1 (ACO1). Electrophoretic mobility shift and chromatin immune-precipitation assays showed that the BR transcription factor BES1 directly binds to two E-box sequences located in the intergenic region of ACO1. GUS expression using site mutations of the E-box sequences verified that ACO1 is normally expressed only when BES1 binds to the E-boxes in the putative promoter of ACO1, indicating that this binding is essential for ACO1 expression and the subsequent production of ethylene in A. thaliana roots. BR exogenously applied to A. thaliana roots enhanced the gravitropic response. Additionally, bes1-D exhibited a greater gravitropic response than did the wild-type specimens, proving that BR is a positive regulator of the gravitropic response in A. thaliana roots. The knock-down mutant aco1-1 showed a slightly lower gravitropic response than did the wild-type specimens, while bes1-D X aco1-1 exhibited a lower gravitropic response than did bes1-D. Therefore, ACO1 is a direct downstream target for BR transcription factor BES1, which controls ethylene production for gravitropism in A. thaliana roots.

Arabidopsis ACC Oxidase 1 Coordinated by Multiple Signals Mediates Ethylene Biosynthesis and Is Involved in Root Development.

Ethylene regulates numerous aspects of plant growth and development. Multiple external and internal factors coordinate ethylene production in plant tissues. Transcriptional and post-translational regulations of ACC synthases (ACSs), which are key enzymes mediating a rate-limiting step in ethylene biosynthesis have been well characterized. However, the regulation and physiological roles of ACC oxidases (ACOs) that catalyze the final step of ethylene biosynthesis are largely unknown in Arabidopsis. Here, we show that Arabidopsis ACO1 exhibits a tissue-specific expression pattern that is regulated by multiple signals, and plays roles in the lateral root development in Arabidopsis. Histochemical analysis of the ACO1 promoter indicated that ACO1 expression was largely modulated by light and plant hormones in a tissue-specific manner. We demonstrated that point mutations in two E-box motifs on the ACO1 promoter reduce the light-regulated expression patterns of ACO1. The aco1-1 mutant showed reduced ethylene production in root tips compared to wild-type. In addition, aco1-1 displayed altered lateral root formation. Our results suggest that Arabidopsis ACO1 integrates various signals into the ethylene biosynthesis that is required for ACO1's intrinsic roles in root physiology.

OST1 Activation by the Brassinosteroid-Regulated Kinase CDG1-LIKE1 in Stomatal Closure.

Crosstalk between signaling pathways is an important feature of complex regulatory networks. How signal crosstalk circuits are tailored to suit different needs of various cell types remains a mystery in biology. Brassinosteroid (BR) and abscisic acid (ABA) antagonistically regulate many aspects of plant growth and development through direct interactions between components of the two signaling pathways. Here, we show that BR and ABA synergistically regulate stomatal closure through crosstalk between the BR-activated kinase CDG1-LIKE1 (CDL1) and the OPEN STOMATA1 (OST1) of the ABA signaling pathway in Arabidopsis thaliana We demonstrate that the cdl1 mutant displayed reduced sensitivity to ABA in a stomatal closure assay, similar to the ost1 mutant. CDL1 and the BR receptor BR-INSENSITIVE1, but not other downstream components of the BR signaling pathway, were required for BR regulation of stomatal movement. Genetic and biochemical experiments demonstrated that CDL1 activates OST1 by phosphorylating it on residue Ser-7. BR increased phosphorylation of OST1, and the BR-induced OST1 activation was abolished in cdl1 mutants. Moreover, we found that ABA activates CDL1 in an OST1-dependent manner. Taken together, our findings illustrate a cell-type-specific BR signaling branch through which BR acts synergistically with ABA in regulating stomatal closure.

Function and molecular regulation of DWARF1 as a C-24 reductase in brassinosteroid biosynthesis in Arabidopsis.

DWARF1 (DWF1) is a sterol C-24 reductase that catalyses the conversion of 24-methylenecholesterol (24-MCHR) to campesterol (CR) in Arabidopsis. A loss-of-function mutant, dwf1, showed similar phenotypic abnormalities to brassinosteroid (BR)-deficient mutants. These abnormalities were reversed in the wild-type phenotype by exogenous application of castasterone (CS) and brassinolide (BL), but not dolichosterone (DS). Accumulation of DS and decreased CS were found in quantitative analysis of endogenous BRs in dwf1. The enzyme solution prepared from dwf1 was unable to convert 6-deoxoDS to 6-deoxoCS and DS to CS, as seen in either wild-type or 35S:DWF1 transgenic plants. This suggests that DWF1 has enzyme activity not only for a sterol C-24 reductase, but also for a BR C-24 reductase that catalyses C-24 reduction of 6-deoxoDS to 6-deoxoCS and of DS to CS in Arabidopsis. Overexpression of DWF1 in a BR-deficient mutant (det2 35S:DWF1) clearly rescued abnormalities found in det2, indicating that DWF1 functions in biosynthesis of active BRs in Arabidopsis. Expression of DWF1 is down-regulated by application of CS and BL and in a BR-dominant mutant, bes1-D. E-boxes in the putative promoter region of DWF1 directly bind to a BR transcription factor, BES1, implying that DWF1 expression is feedback-regulated by BR signaling via BES1. Overall, biosynthesis of 24-methylene BR is an alternative route for generating CS, which is mediated and regulated by DWF1 in Arabidopsis.

ARF7 increases the endogenous contents of castasterone through suppression of BAS1 expression in Arabidopsis thaliana.

Homeostasis of brassinosteroids (BRs) maintained by the balance between their biosynthesis and inactivation is important to coordinate the diverse physiological and developmental responses of plants. Although BR signaling regulates the endogenous levels of BRs via negative feedback regulation, it remains largely unknown how the biosynthesis and inactivation of BR are triggered. BAS1 encodes CYP734A1, which inactivates the biologically active BRs via C-26 hydroxylation and is down-regulated by a BR-responsive transcription factor, BZR1. Here it is demonstrated that the expression of the BAS1 gene is regulated by auxin response factors (ARFs) in Arabidopsis thaliana. Two successive E-box motifs on the BAS1 promoter function as BZR1 binding sites and are essential for BR-regulated BAS1 expression. The expression of BAS1 is increased in the arf7 and arf7arf19 mutants. The endogenous level of bioactive BR, castasterone, is greatly decreased in those mutants. ARF7 can bind to the E-box motifs of the BAS1 promoter where BZR1 binds, suggesting that ARF7 and BZR1 mutually compete for the same cis-element of the BAS1 promoter. Additionally, ARF7 directly interacts with BZR1, which inhibits their DNA binding activities and regulation of BAS1 expression. In conclusion, auxin signaling via ARF7 directly modulates the expression of BAS1 by competition with BZR1, thereby increasing the level of castasterone and promoting growth and development in A. thaliana.

Functional insights of plant GSK3-like kinases: multi-taskers in diverse cellular signal transduction pathways.

The physiological importance of GSK3-like kinases in plants emerged when the functional role of plant GSK3-like kinases represented by BIN2 was first elucidated in the brassinosteroid (BR)-regulated signal transduction pathway. While early studies focused more on understanding how GSK3-like kinases regulate BR signaling, recent studies have implicated many novel substrates of GSK3-like kinases that are involved in a variety of cellular processes as well as BR signaling. Plant GSK3-like kinases play diverse roles in physiological and developmental processes such as cell growth, root and stomatal cell development, flower development, xylem differentiation, light response, and stress responses. Here, we review the progress made in recent years in understanding the versatile functions of plant GSK3-like kinases. Based on the relationship between GSK3-like kinases and their newly identified substrates, we discuss the physiological and biochemical relevance of various cellular signaling mediated by GSK3-like kinases in plants.

Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum.

An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC-MS analysis in both plants. In contrast, the polar fractions obtained from BR-deficient mutants, Arabidopsis cyp85a2 and tomato d(x) , did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants.

Antagonistic regulation of growth and immunity by the Arabidopsis basic helix-loop-helix transcription factor homolog of brassinosteroid enhanced expression2 interacting with increased leaf inclination1 binding bHLH1.

Plants need to finely balance resources allocated to growth and immunity to achieve optimal fitness. A tradeoff between pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and brassinosteroid (BR)-mediated growth was recently reported, but more information about the underlying mechanisms is needed. Here, we identify the basic helix-loop-helix (bHLH) transcription factor homolog of brassinosteroid enhanced expression2 interacting with IBH1 (HBI1) as a negative regulator of PTI signaling in Arabidopsis (Arabidopsis thaliana). HBI1 expression is down-regulated in response to different PAMPs. HBI1 overexpression leads to reduced PAMP-triggered responses. This inhibition correlates with reduced steady-state expression of immune marker genes, leading to increased susceptibility to the bacterium Pseudomonas syringae. Overexpression of the HBI1-related bHLHs brassinosteroid enhanced expression2 (BEE2) and cryptochrome-interacting bHLH (CIB1) partially inhibits immunity, indicating that BEE2 and CIB1 may act redundantly with HBI1. In contrast to its expression pattern upon PAMP treatment, HBI1 expression is enhanced by BR treatment. Also, HBI1-overexpressing plants are hyperresponsive to BR and more resistant to the BR biosynthetic inhibitor brassinazole. HBI1 is nucleus localized, and a mutation in a conserved leucine residue within the first helix of the protein interaction domain impairs its function in BR signaling. Interestingly, HBI1 interacts with several inhibitory atypical bHLHs, which likely keep HBI1 under negative control. Hence, HBI1 is a positive regulator of BR-triggered responses, and the negative effect of PTI is likely due to the antagonism between BR and PTI signaling. This study identifies a novel component involved in the complex tradeoff between innate immunity and BR-regulated growth.