Brassinosteroid coordinates cell layer interactions in plants via cell wall and tissue mechanics.

Growth coordination between cell layers is essential for development of most multicellular organisms. Coordination may be mediated by molecular signaling and/or mechanical connectivity between cells, but how genes modify mechanical interactions between layers is unknown. Here we show that genes driving brassinosteroid synthesis promote growth of internal tissue, at least in part, by reducing mechanical epidermal constraint. We identified a brassinosteroid-deficient dwarf mutant in the aquatic plant Utricularia gibba with twisted internal tissue, likely caused by mechanical constraint from a slow-growing epidermis. We tested this hypothesis by showing that a brassinosteroid mutant in Arabidopsis enhances epidermal crack formation, indicative of increased tissue stress. We propose that by remodeling cell walls, brassinosteroids reduce epidermal constraint, showing how genes can control growth coordination between layers by means of mechanics.

Wheat Type One Protein Phosphatase Participates in the Brassinosteroid Control of Root Growth via Activation of BES1.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

Local brassinosteroid biosynthesis enables optimal root growth.

Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

Identification of a putative candidate gene encoding 7-dehydrocholesterol reductase involved in brassinosteroids biosynthesis for compact plant architecture in Cucumber (Cucumis sativus L.).

KEY MESSAGE: By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, which encodes 7 dehydrocholesterol reductase that involved in brassinosteroids biosynthesis, was identified as the candidate gene for cpa. Dwarf architecture is one of the most important breeding goals in crops. The biosynthesis and signal transduction of brassinosteroids (BRs) have a great impact on plant growth and development including plant architecture. Here, we identified a compact plant architecture (cpa) mutant from an EMS-induced cucumber population. cpa displayed the extremely dwarf phenotype with shortened internode and petiole, darkened and wrinkled leaf. Genetic analysis revealed that cpa was caused by a single recessive gene. By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, encoding a 7-dehydrocholesterol reductase that involved in sterol biosynthesis, was identified as the candidate gene for cpa. One single nucleotide mutation (G→A) in splicing site causing 3-bp insertion (TAG) was found in the first base of the sixth intron of CsDWF5 in cpa, which furtherly resulted in the frameshift mutation and got a premature stop codon. The expression of CsDWF5 gene was significantly down regulated in different tissues of the cpa mutant compared with that in wild type. The phenotype of cpa could be partially recovered by exogenous BR treatment. Transcriptome analysis identified 1096 genes that exhibited differential expression between the cpa mutant and wild type. KEGG enrichment analysis indicated that differentially expressed genes were significantly enriched in BR biosynthesis and plant-pathogen interaction pathways. These results provide perspectives on the molecular mechanisms underlying the dwarfing phenotype in cucumber.

Suppression of Rice Cryptochrome 1b Decreases Both Melatonin and Expression of Brassinosteroid Biosynthetic Genes Resulting in Salt Tolerance.

We investigated the relationship between the blue-light photoreceptor cryptochrome (CRY) and melatonin biosynthesis by generating RNA interference (RNAi) transgenic rice plants that suppress the cryptochrome 1b gene (CRY1b). The resulting CRY1b RNAi rice lines expressed less CRY1b mRNA, but not CRY1a or CRY2 mRNA, suggesting that the suppression is specific to CRY1b. The growth of CRY1b RNAi rice seedlings was enhanced under blue light compared to wild-type growth, providing phenotypic evidence for impaired CRY function. When these CRY1b RNAi rice plants were challenged with cadmium to induce melatonin, wild-type plants produced 100 ng/g fresh weight (FW) melatonin, whereas CRY1b RNAi lines produced 60 ng/g FW melatonin on average, indicating that melatonin biosynthesis requires the CRY photoreceptor. Due to possible feedback regulation, the expression of melatonin biosynthesis genes such as T5H, SNAT1, SNAT2, and COMT was elevated in the CRY1b RNAi lines compared to the wild-type plants. In addition, laminar angles decreased in the CRY1b RNAi lines via the suppression of brassinosteroid (BR) biosynthesis genes such as DWARF. The main cause of the BR decrease in the CRY1b RNAi lines seems to be the suppression of CRY rather than decreased melatonin because the melatonin decrease suppressed DWARF4 rather than DWARF.

The brassinosteroid biosynthesis gene, ZmD11, increases seed size and quality in rice and maize.

Brassinosteroids (BRs) are a group of plant steroid hormones that regulate many important agronomic traits. Studies on the functional mechanisms of BR-related genes in crop plants are necessary for the application of BRs in agriculture. In this study, ZmD11, an ortholog of rice DWARF11 (D11), and 42 other BR biosynthesis-related genes were identified in maize (Zea mays). Complementary experiments confirmed that ZmD11 completely rescued the abnormal panicle architecture and plant height of the rice cpb1 mutant. A phylogenetic analysis indicated that ZmD11-like proteins were found in other monocots and dicots, but not in lower plants and that alternative splicing variants of these homologues mainly exist in Triticeae crops. A subcellular localization analysis showed that ZmD11 localized to the endoplasmic reticulum. The ZmD11 gene was predominantly expressed in young ears and seeds from 10 to 16 days after pollination, especially in the scutellar aleurone layer and pericarp. Furthermore, the constitutive expression of the ZmD11 gene significantly increased seed length, seed area, seed weight and both seed starch and protein contents in rice and maize. Our results suggest that ZmD11 is a key gene in the regulation of seed size and quality and that it has a potential application value in the molecular breeding of crops.

Brassinosteroids synthesised by CYP85A/A1 but not CYP85A2 function via a BRI1-like receptor but not via BRI1 in Picea abies.

Brassinosteroids (BRs) are essential plant hormones. In angiosperms, brassinolide and castasterone, the first and second most active BRs, respectively, are synthesised by CYP85A2 and CYP85A/A1, respectively. BRs in angiosperms function through an essential receptor, BR Insensitive 1 (BRI1). In addition, some angiosperms also have non-essential BRI1-like 1/3 (BRL1/3). In conifers, BRs promote seed germination under drought stress; however, how BRs function in gymnosperms is unknown. In this study, we performed functional complementation of BR biosynthesis and receptor genes from Picea abies with respective Arabidopsis mutants. We found that P. abies possessed functional PaCYP85A and PaBRL1 but not PaCYP85A2 or PaBRI1, and this results in weak BR signaling, and both PaCYP85A and PaBRL1 were abundantly expressed. However, neither BR treatment of P. abies seedlings nor expression of PaBRL1 in the Arabidopsis Atbri1 mutant promoted plant height, despite the fact that BR-responsive genes were activated. Importantly, chimeric AtBRI1 replaced with the BR-binding domain of PaBRL1 complemented the Atbri1 phenotypes. Furthermore, PaBRL1 had less kinase activity than BRI1 in vitro. Overall, P. abies had weak but still active BR signaling, explaining aspects of its slow growth and high stress tolerance. Our study sheds light on the functional and evolutionary significance of distinct BR signaling that is independent of BRI1 and brassinolide.

Phytochrome A inhibits shade avoidance responses under strong shade through repressing the brassinosteroid pathway in Arabidopsis.

In dense canopy, a reduction in red to far-red (R/FR) light ratio triggers shade avoidance responses (SARs) in Arabidopsis thaliana, a shade avoiding plant. Two red/far-red (R/FR) light photoreceptors, PHYB and PHYA, were reported to be key negative regulators of the SARs. PHYB represses the SARs under normal light conditions; however, the role of PHYA in the SARs remains elusive. We set up two shade conditions: Shade and strong Shade (s-Shade) with different R/FR ratios (0.7 and 0.1), which allowed us to observe phenotypes dominated by PHYB- and PHYA-mediated pathway, respectively. By comparing the hypocotyl growth under these two conditions with time, we found PHYA was predominantly activated in the s-Shade after prolonged shade treatment. We further showed that under s-Shade, PHYA inhibits hypocotyl elongation partially through repressing the brassinosteroid (BR) pathway. COP1 and PIF4,5 act downstream of PHYA. After prolonged shade treatment, the nuclear localization of COP1 was reduced, while the PIF4 protein level was much lower in the s-Shade than that in Shade. Both changes occurred in a PHYA-dependent manner. We propose that under deep canopy, the R/FR ratio is extremely low, which promotes the nuclear accumulation of PHYA. Activated PHYA reduces COP1 nuclear speckle, which may lead to changes of downstream targets, such as PIF4,5 and HY5. Together, these proteins regulate the BR pathway through modulating BES1/BZR1 and the expression of BR biosynthesis and BR target genes.

Comparative phosphoproteomic analysis of BR-defective mutant reveals a key role of GhSK13 in regulating cotton fiber development.

Brassinosteroid (BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1 (pag1), and its corresponding wild-type (ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage (10 days after pollination (DPA)) and secondary cell wall synthesis stage (20 DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs (GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor Gh_DNF_YB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.

Obtusifoliol 14α-demethylase OsCYP51G1 is involved in phytosterol synthesis and affects pollen and seed development.

As structural components of biological membranes, phytosterols are essential not only for a variety of cellular functions but are also precursors for brassinosteroid (BR) biosynthesis. Plant CYP51 is the oldest and most conserved obtusifoliol 14α-demethylase in eukaryotes and is an essential component of the sterol biosynthesis pathway. However, little is known about rice (Oryza sativa L.) CYP51G1. In this study, we showed that rice OsCYP51G1 shared high homology with obtusifoliol 14α-demethylase and OsCYP51G1 was strongly expressed in most of rice organs. Subcellular localization analysis indicated that OsCYP51G1 was localized to the endoplasmic reticulum. Knockdown and knockout of OsCYP51G1 resulted in delayed flowering, impaired membrane integrity, abnormal pollen, and reduced grain yield, whereas OsCYP51G1 overexpression led to increased grain yield. Knockdown of OsCYP51G1 also reduced the levels of end-products (sitosterol and stigmasterol) and increased those of upstream intermediates (24-methylene-cycloartenol and cycloeucalenol) of the OsCYP51G1-mediated sterol biosynthesis step. In contrast, overexpression of OsCYP51G1 increased the sitosterol and stigmasterol content and reduced that of cycloeucalenol. However, knockdown of OsCYP51G1 by RNAi did not elicit these BR deficiency-related phenotypes, such as dwarfism, erect leaves and small seeds, nor was the leaf lamina angle sensitive to brassinolide treatment. These results revealed that rice OsCYP15G1 encodes an obtusifoliol 14α-demethylase for the phytosterols biosynthesis and possible without affecting the biosynthesis of downstream BRs, which was different from its homolog, OsCYP51G3.

The Root Foraging Response under Low Nitrogen Depends on DWARF1-Mediated Brassinosteroid Biosynthesis.

Root developmental plasticity enables plants to adapt to limiting or fluctuating nutrient conditions in the soil. When grown under nitrogen (N) deficiency, plants develop a more exploratory root system by increasing primary and lateral root length. However, mechanisms underlying this so-called foraging response remain poorly understood. We performed a genome-wide association study in Arabidopsis (Arabidopsis thaliana) and we show here that noncoding variations of the brassinosteroid (BR) biosynthesis gene DWARF1 (DWF1) lead to variation of the DWF1 transcript level that contributes to natural variation of root elongation under low N. In addition to DWF1, other central BR biosynthesis genes upregulated under low N include CONSTITUTIVE PHOTOMORPHOGENIC DWARF, DWF4, and BRASSINOSTEROID-6-OXIDASE 2 Phenotypic characterization of knockout and knockdown mutants of these genes showed significant reduction of their root elongation response to low N, suggesting a systemic stimulation of BR biosynthesis to promote root elongation. Moreover, we show that low N-induced root elongation is associated with aboveground N content and that overexpression of DWF1 significantly improves plant growth and overall N accumulation. Our study reveals that mild N deficiency induces key genes in BR biosynthesis and that natural variation in BR synthesis contributes to the root foraging response, complementing the impact of enhanced BR signaling observed recently. Furthermore, these results suggest a considerable potential of BR biosynthesis to genetically engineer plants with improved N uptake.

Molecular Mechanisms of Brassinosteroid-Mediated Responses to Changing Environments in Arabidopsis.

Plant adaptations to changing environments rely on integrating external stimuli into internal responses. Brassinosteroids (BRs), a group of growth-promoting phytohormones, have been reported to act as signal molecules mediating these processes. BRs are perceived by cell surface receptor complex including receptor BRI1 and coreceptor BAK1, which subsequently triggers a signaling cascade that leads to inhibition of BIN2 and activation of BES1/BZR1 transcription factors. BES1/BZR1 can directly regulate the expression of thousands of downstream responsive genes. Recent studies in the model plant Arabidopsis demonstrated that BR biosynthesis and signal transduction, especially the regulatory components BIN2 and BES1/BZR1, are finely tuned by various environmental cues. Here, we summarize these research updates and give a comprehensive review of how BR biosynthesis and signaling are modulated by changing environments and how these changes regulate plant adaptive growth or stress tolerance.

Light Activates Brassinosteroid Biosynthesis to Promote Hook Opening and Petiole Development in Arabidopsis thaliana.

Although brassinosteroids (BRs) have been proposed to be negative regulators of photomorphogenesis, their physiological role therein has remained elusive. We studied light-induced photomorphogenic development in the presence of the BR biosynthesis inhibitor, brassinazole (Brz). Hook opening was inhibited in the presence of Brz; this inhibition was reversed in the presence of brassinolide (BL). Hook opening was accompanied by cell expansion on the inner (concave) side of the hook. This cell expansion was inhibited in the presence of Brz but was restored upon the addition of BL. We then evaluated light-induced organ-specific expression of three BR biosynthesis genes, DWF4, BR6ox1 and BR6ox2, and a BR-responsive gene, SAUR-AC1, during the photomorphogenesis of Arabidopsis. Expression of these genes was induced, particularly in the hook region, in response to illumination. The induction peaked after 3 h of light exposure and preceded hook opening. Phytochrome-deficient mutants, hy1, hy2 and phyAphyB, and a light-signaling mutant, hy5, were defective in light-induced expression of BR6ox1, BR6ox2 and SAUR-AC1. Light induced both expression of BR6ox genes and petiole development. Petiole development was inhibited in the presence of Brz. Our results largely contradict the early view that BRs are negative regulators of photomorphogenesis. Our data collectively suggest that light activates the expression of BR biosynthesis genes in the hook region via a phytochrome-signaling pathway and HY5 and that BR biosynthesis is essential for hook opening and petiole development during photomorphogenesis.

PcDWF1, a pear brassinosteroid biosynthetic gene homologous to AtDWARF1, affected the vegetative and reproductive growth of plants.

BACKGROUND: The steroidal hormones brassinosteroids (BRs) play important roles in plant growth and development. The pathway and genes involved in BR biosynthesis have been identified primarily in model plants like Arabidopsis, but little is known about BR biosynthesis in woody fruits such as pear. RESULTS: In this study, we found that applying exogenous brassinolide (BL) could significantly increase the stem growth and rooting ability of Pyrus ussuriensis. PcDWF1, which had a significantly lower level of expression in the dwarf-type pear than in the standard-type pear, was cloned for further analysis. A phylogenetic analysis showed that PcDWF1 was a pear brassinosteroid biosynthetic gene that was homologous to AtDWARF1. The subcellular localization analysis indicated that PcDWF1 was located in the plasma membrane. Overexpression of PcDWF1 in tobacco (Nicotiana tabacum) or pear (Pyrus ussuriensis) plants promoted the growth of the stems, which was caused by a larger cell size and more developed xylem than those in the control plants, and the rooting ability was significantly enhanced. In addition to the change in vegetative growth, the tobacco plants overexpressing PcDWF1 also had a delayed flowering time and larger seed size than did the control tobacco plants. These phenotypes were considered to result from the higher BL contents in the transgenic lines than in the control tobacco and pear plants. CONCLUSIONS: Taken together, these results reveal that the pear BR biosynthetic gene PcDWF1 affected the vegetative and reproductive growth of Pyrus ussuriensis and Nicotiana tabacum and could be characterized as an important BR biosynthetic gene in perennial woody fruit plants.

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.

HSP Transcript and Protein Accumulation in Brassinosteroid Barley Mutants Acclimated to Low and High Temperatures.

In temperature stress, the main role of heat-shock proteins (HSP) is to act as molecular chaperones for other cellular proteins. However, knowledge about the hormonal regulation of the production of the HSP is quite limited. Specifically, little is known about the role of the plant steroid hormones-brassinosteroids (BR)-in regulating the HSP expression. The aim of our study was to answer the question of how a BR deficit or disturbances in its signaling affect the accumulation of the HSP90, HSP70, HSP18, and HSP17 transcripts and protein in barley growing at 20 °C (control) and during the acclimation of plants at 5 °C and 27 °C. In barley, the temperature of plant growth modified the expression of HSPs. Furthermore, the BR-deficient mutants (mutations in the HvDWARF or HvCPD genes) and BR-signaling mutants (mutation in the HvBRI1 gene) were characterized by altered levels of the transcripts and proteins of the HSP group compared to the wild type. The BR-signaling mutant was characterized by a decreased level of the HSP transcripts and heat-shock proteins. In the BR-deficient mutants, there were temperature-dependent cases when the decreased accumulation of the HSP70 and HSP90 transcripts was connected to an increased accumulation of these HSP. The significance of changes in the accumulation of HSPs during acclimation at 27 °C and 5 °C is discussed in the context of the altered tolerance to more extreme temperatures of the studied mutants (i.e., heat stress and frost, respectively).

14-3-3 proteins contribute to leaf and root development via brassinosteroid insensitive 1 in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BR) are essential growth hormone in plants. Various components involved in signal transduction pathway have been identified as targets of 14-3-3 phospho-binding proteins. Previously, we showed that 14-3-3 proteins directly interact with the Brassinosteroid Insensitive 1 (BRI1), the BR receptor kinase, and are also subject to phosphorylation in a BR-dependent manner. OBJECTIVE: In this study, we aimed to examine a potential interplay between 14-3-3 proteins and BRI1 in plant growth. METHODS: Morphological phenotypes of a T-DNA insertion mutant line, 14-3-3ψφε, defective in three 14-3-3 isoforms, psi, phi and epsilon, were characterized and compared with bri1-5 and two transgenic lines for BRI1, BRI1-Flag and BRI1-Flag (14-3-3ψφε). We also generated complementation lines carrying each of the three 14-3-3 genes and determined their differences in rosette growth. RESULTS: No significant differences between the wild-type and 14-3-3ψφε seedlings were observed regardless of BR applications. However, BRI1-Flag (14-3-3ψφε) showed a significantly reduced cold tolerance and BR sensitivity in hypocotyl and root development when compared to BRI1-Flag. In addition, narrower leaf shape and smaller rosette size were observed in BRI1-Flag (14-3-3ψφε), while the mutant phenotypes were partially restored in the complementation lines, two of which with 14-3-3φ and 14-3-3ε showed the rosette growth comparable to BRI1-Flag. CONCLUSION: Taken together, our results suggested that 14-3-3 proteins might positively regulate BRI1 activity and showed that 14-3-3 isoforms have different functional impacts in BR signaling.

Brassinosteroids Antagonize Jasmonate-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1).

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

Inhibitors of Brassinosteroid Biosynthesis and Signal Transduction.

Chemical inhibitors are invaluable tools for investigating protein function in reverse genetic approaches. Their application bears many advantages over mutant generation and characterization. Inhibitors can overcome functional redundancy, their application is not limited to species for which tools of molecular genetics are available and they can be applied to specific tissues or developmental stages, making them highly convenient for addressing biological questions. The use of inhibitors has helped to elucidate hormone biosynthesis and signaling pathways and here we review compounds that were developed for the plant hormones brassinosteroids (BRs). BRs are steroids that have strong growth-promoting capacities, are crucial for all stages of plant development and participate in adaptive growth processes and stress response reactions. In the last two decades, impressive progress has been made in BR inhibitor development and application, which has been instrumental for studying BR modes of activity and identifying and characterizing key players. Both, inhibitors that target biosynthesis, such as brassinazole, and inhibitors that target signaling, such as bikinin, exist and in a comprehensive overview we summarize knowledge and methodology that enabled their design and key findings of their use. In addition, the potential of BR inhibitors for commercial application in plant production is discussed.

Proteome and Ubiquitome Changes during Rose Petal Senescence.

Petal senescence involves numerous programmed changes in biological and biochemical processes. Ubiquitination plays a critical role in protein degradation, a hallmark of organ senescence. Therefore, we investigated changes in the proteome and ubiquitome of senescing rose (Rosa hybrida) petals to better understand their involvement in petal senescence. Of 3859 proteins quantified in senescing petals, 1198 were upregulated, and 726 were downregulated during senescence. We identified 2208 ubiquitinated sites, including 384 with increased ubiquitination in 298 proteins and 1035 with decreased ubiquitination in 674 proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that proteins related to peptidases in proteolysis and autophagy pathways were enriched in the proteome, suggesting that protein degradation and autophagy play important roles in petal senescence. In addition, many transporter proteins accumulated in senescing petals, and several transport processes were enriched in the ubiquitome, indicating that transport of substances is associated with petal senescence and regulated by ubiquitination. Moreover, several components of the brassinosteroid (BR) biosynthesis and signaling pathways were significantly altered at the protein and ubiquitination levels, implying that BR plays an important role in petal senescence. Our data provide a comprehensive view of rose petal senescence at the posttranslational level.