Quantitative reverse transcription PCR (qRT-PCR) analysis and ProACO2::GUS expression showed that ACO2 was highly expressed in the shoots of Arabidopsis seedlings under light conditions. Exogenously applied aminocyclopropane-1-carboxylic acid (ACC) enhanced the expression of ACO2, whereas Co2+ ions suppressed its expression. In comparison with wild-type seedlings, the ACO2 knockdown mutant aco2-1 produced less ethylene, which resulted in the inhibited growth of Arabidopsis seedlings. Exogenously applied brassinolide reduced the expression of ACO2. ACO2 expression was increased in det2, a brassinosteroid (BR)-deficient mutant; however, it was decreased in bes1-D, a brassinosteroid insensitive 1-EMS-suppressor 1 (BES1)-dominant mutant. In the putative promoter region of ACO2, 11 E-box sequences for BES1 binding but not BR regulatory element sequences for brassinazole-resistant 1 (BZR1) binding were found. Chromatin immunoprecipitation assay showed that BES1 could directly bind to the E-boxes located in the putative promoter region of ACO4. Less ethylene was produced in bes1-D seedlings compared with wild-type seedlings, suggesting that the direct binding of BES1 to the ACO2 promoter may negatively regulate ACO2 expression to control the endogenous level of ethylene in Arabidopsis seedlings.

The increased level of endogenous abscisic acid (ABA) in brassinosteroid (BR)-deficient mutants, such as det2 and cyp85a1 x 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 x 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 x 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.

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.

Arabidopsis Cytochrome P450 85A2 (AtCYP85A2) was introduced to Arabidopsis thaliana seeds using a seed-specific promoter, pAt5g54000. GUS (beta-Glucuronidase) activity and RT-PCR analysis demonstrated that AtCYP85A2 overexpression only occurred in seeds of a transgenic plant, pAt5g54000-AtCYP85A2::Col-0. A crude enzyme solution prepared pAt5g54000-AtCYP85A2::Col-0 seeds successfully catalyzed the conversion of castasterone (CS) to brassinolide (BL), which was not detected in wild-type seeds. Furthermore, a higher level of CS and BL was detected in pAt5g54000-AtCYP85A2::Col-0 seeds compared to untransformed seeds, thus demonstrating that seed-specific overexpression of AtCYP85A2 efficiently encoded a bi-functional enzyme for brassinosteroids 6-oxidase/brassinolide synthase to generate CS and BL in seeds of pAt5g54000-AtCYP85A2::Col-0. Compared to the wild type, pAt5g54000-AtCYP85A2::Col-0 produced substantially larger seeds with a high concentration of nutrients due to an enhancement in brassinosteroids signaling. Additionally, pAt5g54000-AtCYP85A2::Col-0 exhibited superior seed germination, seedling and rosette plant growth, and flower and silique formation, indicating that seed-specific AtCYP85A2 expression activates overall vegetative and reproductive growth and development in A. thaliana.

Brassinosteroid regulates carotenoid content through BZR1-mediated suppression of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASEin Arabidopsis seedlings.

The crosstalk mechanism regulating content and signal transduction between brassinosteroids (BRs) and salicylic acid (SA) for plant defense was investigated in Arabidopsis. Compared to the wild type, an increased bacterial resistance was observed in bzr1-1D, a dominant mutant of the BR transcription factor BZR1. In bzr1-1D, SA biosynthetic gene ICS1 expression and endogenous SA content greatly increased upon Pst DC3000 infection, and the direct binding of BZR1 to the ICS1 promoter was confirmed through EMSA and ChIP. In bzr1-1D where NPR1 expression was almost absent, expression of PR genes was increased, and both BZR1 and PR5 expressions increased after SA treatment. EMSA and ChIP verified that BZR1 binds directly to the cis-element present in the PR5 promoter and a pull-down assay showed that TGAs, SA transcription factors upstream of PR genes, interact with BZR1 at the protein level. Crude enzyme assays demonstrated that BR C-6 oxidase activity, a CYP85A1 function, greatly increased during Pst DC3000 infection. In the tga1 tga4 double mutant lacking SA transcription factors TGA1 and TGA4, BR biosynthetic gene CYP85A1 expression was significantly reduced. EMSA and ChIP confirmed that both TGA1 and TGA4 bind to the cis-element present in the CYP85A1 promoter, and castasterone (CS), a bio-active BR, was significantly reduced in tga1 tga4. Taken together, the upregulation of ICS1 expression by BZR1 and CYP85A1 expression by TGA1/4 mutually enhanced endogenous level of BR and SA in Arabidopsis. Furthermore, TGAs and BZR1 interaction at the protein level induces SA-induced immunity through the upregulation of PR5 expression, increasing bacterial resistance in the plant. These results explain the mutual control mechanisms of the synergistic effects BR and SA have on plant defense and confirm BR's effect on plant defense and growth promotion in A. thaliana.

This study generated the transgenic Arabidopsis lines pAt5g54000-AtBZR1::Col-0 and pAt5g54000-AtBES1::Col-0, in which Arabidopsis BZR1 and BES1 (AtBZR1 and AtBES1) were seed-specifically expressed with pAt5g54000, a seed-specific promoter. Semi-quantitative RT-PCR and GUS-staining analysis demonstrated that the inserted AtBZR1 and AtBES1 were concentrated in seeds in siliques of transgenic plants. Seed number, length, width, and mass increased in the pAt5g54000-AtBZR1::Col-0 and pAt5g54000-AtBES1::Col-0 mutants compared to untransformed Arabidopsis. The endogenous levels of primary metabolites, such as carbohydrates, proteins, and lipids, in transgenic seeds were also higher than those in wild-type seeds, indicating that both seed size and quality are improved by seed-specific expression of AtBZR1 and AtBES1 in Arabidopsis. In both transgenic Arabidopsis seeds, relative to wild-type seeds, the expression of positive regulatory genes involved in determining seed size, such as SHORT HYPOCOTYL UNDER BLUE1 (AtSHB1), MINISEED3 (AtMINI3), and HAIKU2 (AtIKU2), was increased by up-regulation of AtBZR1 and AtBES1 as well as down-regulation of ABA Deficient 2 (AtABA2) and ABA Insensitive 5 (AtABI5). This result suggests that AtBZR1- and AtBES1-mediated signaling pathways such as AtBZR1/AtBES1 -> AtSHB1 -> AtMINI3 -> AtIKU2 and AtBZR1/AtBES1 -> AtABA2, and/or AtABI5 -> AtSHB1 -> AtMINI3 -> AtIKU2 increase the yield and quality of seeds in transgenic Arabidopsis. Taken together, our findings demonstrated the usefulness and applicability of seed-specific introduction of AtBZR1 and AtBES1 encoding key transcription factors in brassinosteroid signaling to improve seed yield and quality in Arabidopsis.

Expression of ABA-deficient 2 (ABA2) gene involved in ABA biosynthesis was downregulated in bes1-D, but upregulated in bes1-KO. Hence, the expression of ABA2 is negatively controlled by the BES1 transcription factor of brassinosteroid signaling in Arabidopsis thaliana. BES1 is directly bound to the E-box sequences in the promoter of ABA2, which reduced the endogenous levels of ABA in the plant. The seeds of aba2-1 and bes1-KO were larger and smaller, respectively, than those of the wild type. In bes1-KO x aba2-1, the reduced seed size in bes1-KO was partly restored to the seed size in aba2-1. Hence, BES1-mediated regulation of seed size is an upstream process for the homeostasis of endogenous ABA to control the seed size. BES1 suppressed the expression of ABA Insensitive 5 (ABI5), a major transcription factor gene in ABA signaling that determines the seed size. It is directly bound to the promoter of ABI5 by BES1-induced downregulation of ABI5. The expression of SHORT HYPOCOTYL UNDER BLUE1, MINISEED3, and HAIKU2 as positively regulatory genes determining the seed size was activated, increasing the seed size in bes1-D. Conclusively, brassinosteroid signaling through BES1 downregulated both the biosynthesis and signaling of ABA, which increased the seed size in Arabidopsis.

Dust grains absorb half of the radiation emitted by stars throughout the history of the universe, re-emitting this energy at infrared wavelengths(1-3). Polycyclic aromatic hydrocarbons (PAHs) are large organic molecules that trace millimetre-size dust grains and regulate the cooling of interstellar gas within galaxies(4,5). Observations of PAH features in very distant galaxies have been difficult owing to the limited sensitivity and wavelength coverage of previous infrared telescopes(6,7). Here we present James Webb Space Telescope observations that detect the 3.3 mu m PAH feature in a galaxy observed less than 1.5 billion years after the Big Bang. The high equivalent width of the PAH feature indicates that star formation, rather than black hole accretion, dominates infrared emission throughout the galaxy. The light from PAH molecules, hot dust and large dust grains and stars are spatially distinct from one another, leading to order-of-magnitude variations in PAH equivalent width and ratio of PAH to total infrared luminosity across the galaxy. The spatial variations we observe suggest either a physical offset between PAHs and large dust grains or wide variations in the local ultraviolet radiation field. Our observations demonstrate that differences in emission from PAH molecules and large dust grains are a complex result of localized processes within early galaxies.