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.

Inhibition of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis.

Unlike the indispensable function of the steroid hormone brassinosteroid (BR) in regulating plant growth and development, the metabolism of secondary metabolites regulated by BR is not well known. Here we show that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated higher content of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid content. We demonstrated that BR transcriptionally suppresses 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. We found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the promoter of HPPD, and HPPD suppression by BR was increased in the bzr1-1D gain-of-function mutation. Interestingly, dark-induced HPPD expression did not cause carotenoid accumulation, due to down-regulation of other carotenoid biosynthetic genes in the dark. Our results suggest that BR regulates different physiological responses in dark and light through inhibition of HPPD expression.

Seed-Specific Expression of Arabidopsis AtCYP85A2 Produces Biologically Active Brassinosteroids Such as Castasterone and Brassinolide to Improve Grain Yield and Quality in Seeds of Brachypodium Distachyon.

Brachypodium distachyon is a monocotyledonous model plant that contains castasterone (CS) but no brassinolide (BL) as the end product of brassinosteroids (BR) biosynthesis, indicating dysfunction of BL synthase, which catalyzes the conversion of CS to BL. To increase BR activity, Arabidopsis cytochrome P450 85A2 (AtCYP85A2) encoding BR 6-oxidase/BL synthase was introduced into B. distachyon with the seed-specific promoters pBSU1, pAt5g10120, and pAt5g54000. RT-PCR analysis and GUS activity revealed that AtCYP85A2 was only expressed in the seeds of the transgenic plants pBSU1-AtCYP85A2::Bd21-3, pAt5g10120-AtCYP85A2::Bd21-3, and pAt5g54000-AtCYP85A2::Bd21-3. The crude enzyme prepared from the seeds of these three transgenic plants catalyzed the conversion of 6-deoxoCS to CS. The transgenic plants showed greater specific enzyme activity than the wild-type plant for the conversion of 6-deoxoCS to CS, indicating enhanced BR 6-oxidase activity in the transgenic plants. The enzyme solution also catalyzed the conversion of CS into BL. Additionally, BL was identified from the seeds of transgenic plants, verifying that seed-specific AtCYP85A2 encodes a functional BL synthase to increase BR activity in the seeds of transgenic Brachypodium. In comparison with wild-type Brachypodium, the transgenic plants showed better growth and development during the vegetative growing stage. The flowers of the transgenic plants were remarkably larger, resulting in increments in the number, size, and height of seeds. The total starch, protein, and lipid contents in transgenic plants were higher than those in wild-type plants, indicating that seed-specific expression of AtCYP85A2 improves both grain yield and quality in B. distachyon.

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.

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.

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.

Overexpression of Arabidopsis thaliana brassinosteroid-related acyltransferase 1 gene induces brassinosteroid-deficient phenotypes in creeping bentgrass.

Brassinosteroids (BRs) are naturally occurring steroidal hormones that play diverse roles in various processes during plant growth and development. Thus, genetic manipulation of endogenous BR levels might offer a way of improving the agronomic traits of crops, including plant architecture and stress tolerance. In this study, we produced transgenic creeping bentgrass (Agrostis stolonifera L.) overexpressing a BR-inactivating enzyme, Arabidopsis thaliana BR-related acyltransferase 1 (AtBAT1), which is known to catalyze the conversion of BR intermediates to inactive acylated conjugates. After putative transgenic plants were selected using herbicide resistance assay, genomic integration of the AtBAT1 gene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by northern blot analysis. The transgenic creeping bentgrass plants exhibited BR-deficient phenotypes, including reduced plant height with shortened internodes (i.e., semi-dwarf), reduced leaf growth rates with short, wide, and thick architecture, high chlorophyll contents, decreased numbers of vascular bundles, and large lamina joint bending angles (i.e., erect leaves). Subsequent analyses showed that the transgenic plants had significantly reduced amounts of endogenous BR intermediates, including typhasterol, 6-deoxocastasterone, and castasterone. Moreover, the AtBAT1 transgenic plants displayed drought tolerance as well as delayed senescence. Therefore, the results of the present study demonstrate that overexpression of an Arabidopsis BR-inactivating enzyme can reduce the endogenous levels of BRs in creeping bentgrass resulting in BR-deficient phenotypes, indicating that the AtBAT1 gene from a dicot plant is also functional in the monocot crop.

SLG controls grain size and leaf angle by modulating brassinosteroid homeostasis in rice.

Grain size and leaf angle are two important traits determining grain yield in rice. However, the mechanisms regulating the two traits remain largely unknown. Here, we characterized a rice gain-of-function mutant, slender grain Dominant (slg-D), which exhibited longer and narrower grains and larger leaf angles, similar to plants with elevated brassinosteroid (BR) levels or strengthened BR signaling. The increased cell length is responsible for the mutant phenotypes in slg-D We demonstrated that the phenotype of slg-D is caused by enhanced expression of SLG, a BAHD acyltransferase-like protein gene. SLG is preferentially expressed in young panicles and lamina joints, implying its role in controlling cell growth in those two tissues. slg-D was restored to wild type by treatment with brassinazole, an inhibitor of BR biosynthesis. Overexpression of SLG in d11-2 (deficient in BR synthesis) and d61-1 (deficient in BR signaling) did not change the existing phenotypes. The slg-D plants had elevated BR contents and, accordingly, expression of BR-related genes was changed in a manner similar to BR treatment. Moreover, SLG RNAi plants displayed mild BR-deficient phenotypes including shorter grains, smaller leaf angles, and compact semi-dwarf plant types. The in vitro biochemical assays and transgenic approaches collectively demonstrated that SLG functions as homomers. Taken together, we conclude that SLG is an important regulator in BR homeostasis and that manipulation of SLG expression to an optimal level may provide a way to develop an ideal plant type.

TOR Signaling Promotes Accumulation of BZR1 to Balance Growth with Carbon Availability in Arabidopsis.

For maintenance of cellular homeostasis, the actions of growth-promoting hormones must be attenuated when nutrient and energy become limiting. The molecular mechanisms that coordinate hormone-dependent growth responses with nutrient availability remain poorly understood in plants [1, 2]. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates nutrient and energy signaling to regulate growth and homeostasis in both animals and plants [3-7]. Here, we show that sugar signaling through TOR controls the accumulation of the brassinosteroid (BR)-signaling transcription factor BZR1, which is essential for growth promotion by multiple hormonal and environmental signals [8-11]. Starvation, caused by shifting of light-grown Arabidopsis seedlings into darkness, as well as inhibition of TOR by inducible RNAi, led to plant growth arrest and reduced expression of BR-responsive genes. The growth arrest caused by TOR inactivation was partially recovered by BR treatment and the gain-of-function mutation bzr1-1D, which causes accumulation of active forms of BZR1 [12]. Exogenous sugar promoted BZR1 accumulation and seedling growth, but such sugar effects were largely abolished by inactivation of TOR, whereas the effect of TOR inactivation on BZR1 degradation is abolished by inhibition of autophagy and by the bzr1-1D mutation. These results indicate that cellular starvation leads sequentially to TOR inactivation, autophagy, and BZR1 degradation. Such regulation of BZR1 accumulation by glucose-TOR signaling allows carbon availability to control the growth promotion hormonal programs, ensuring supply-demand balance in plant growth.