Brassinosteroid Biosynthetic Mutant Research Articles

Transcriptional responses to gibberellin in the maize tassel and control by DELLA domain proteins.

The plant hormone gibberellin (GA) impacts plant growth and development differently depending on the developmental context. In the maize (Zea mays) tassel, application of GA alters floral development, resulting in the persistence of pistils. GA signaling is achieved by the GA-dependent turnover of DELLA domain transcription factors, encoded by dwarf8 (d8) and dwarf9 (d9) in maize. The D8-Mpl and D9-1 alleles disrupt GA signaling, resulting in short plants and normal tassel floret development in the presence of excess GA. However, D9-1 mutants are unable to block GA-induced pistil development. Gene expression in developing tassels of D8-Mpl and D9-1 mutants and their wild-type siblings was determined upon excess GA3 and mock treatments. Using GA-sensitive transcripts as reporters of GA signaling, we identified a weak loss of repression under mock conditions in both mutants, with the effect in D9-1 being greater. D9-1 was also less able to repress GA signaling in the presence of excess GA3 . We treated a diverse set of maize inbred lines with excess GA3 and measured the phenotypic consequences on multiple aspects of development (e.g., height and pistil persistence in tassel florets). Genotype affected all GA-regulated phenotypes but there was no correlation between any of the GA-affected phenotypes, indicating that the complexity of the relationship between GA and development extends beyond the two-gene epistasis previously demonstrated for GA and brassinosteroid biosynthetic mutants.

Brassinosteroids facilitate xylem differentiation and wood formation in tomato.

BR signaling pathways facilitate xylem differentiation and wood formation by fine tuning SlBZR1/SlBZR2-mediated gene expression networks involved in plant secondary growth. Brassinosteroid (BR) signaling and BR crosstalk with diverse signaling cues are involved in the pleiotropic regulation of plant growth and development. Recent studies reported the critical roles of BR biosynthesis and signaling in vascular bundle development and plant secondary growth; however, the molecular bases of these roles are unclear. Here, we performed comparative physiological and anatomical analyses of shoot morphological growth in a cultivated wild-type tomato (Solanum lycopersicum cv. BGA) and a BR biosynthetic mutant [Micro Tom (MT)]. We observed that the canonical BR signaling pathway was essential for xylem differentiation and sequential wood formation by facilitating plant secondary growth. The gradual retardation of xylem development phenotypes during shoot vegetative growth in the BR-deficient MT tomato mutant recovered completely in response to exogenous BR treatment or genetic complementation of the BR biosynthetic DWARF (D) gene. By contrast, overexpression of the tomato Glycogen synthase kinase 3 (SlGSK3) or CRISPR-Cas9 (CR)-mediated knockout of the tomato Brassinosteroid-insensitive 1 (SlBRI1) impaired BR signaling and resulted in severely defective xylem differentiation and secondary growth. Genetic modulation of the transcriptional activity of the tomato Brassinazole-resistant 1/2 (SlBZR1/SlBZR2) confirmed the positive roles of BR signaling pathways for xylem differentiation and secondary growth. Our data indicate that BR signaling pathways directly promote xylem differentiation and wood formation by canonical BR-activated SlBZR1/SlBZR2.

BZR1 Mediates Brassinosteroid-Induced Autophagy and Nitrogen Starvation in Tomato.

Autophagy, an innate cellular destructive mechanism, plays crucial roles in plant development and responses to stress. Autophagy is known to be stimulated or suppressed by multiple molecular processes, but the role of phytohormone signaling in autophagy is unclear. Here, we demonstrate that the transcripts of autophagy-related genes (ATGs) and the formation of autophagosomes are triggered by enhanced levels of brassinosteroid (BR). Furthermore, the BR-activated transcription factor brassinazole-resistant1 (BZR1), a positive regulator of the BR signaling pathway, is involved in BR-induced autophagy. Treatment with BR enhanced the formation of autophagosomes and the transcripts of ATGs in BZR1-overexpressing plants, while the effects of BR were compromised in BZR1-silenced plants. Yeast one-hybrid analysis and chromatin immunoprecipitation coupled with quantitative polymerase chain reaction revealed that BZR1 bound to the promoters of ATG2 and ATG6 The BR-induced formation of autophagosomes decreased in ATG2- and ATG6-silenced plants. Moreover, exogenous application of BR enhanced chlorophyll content and autophagosome formation and decreased the accumulation of ubiquitinated proteins under nitrogen starvation. Leaf chlorosis and chlorophyll degradation were exacerbated in BZR1-silenced plants and the BR biosynthetic mutant d^im but were alleviated in BZR1- and BZR1-1D-overexpressing plants under nitrogen starvation. Meanwhile, nitrogen starvation-induced expression of ATGs and autophagosome formation were compromised in both BZR1-silenced and d^im plants but were increased in BZR1- and BZR1-1D-overexpressing plants. Taken together, our results suggest that BZR1-dependent BR signaling up-regulates the expression of ATGs and autophagosome formation, which plays a critical role in the plant response to nitrogen starvation in tomato (Solanum lycopersicum).

Phytohormone inhibitor treatments phenocopy brassinosteroid-gibberellin dwarf mutant interactions in maize.

SummaryPhytohormone biosynthesis produces metabolites with profound effects on plant growth and development. Modulation of hormone levels during developmental events, in response to the environment, by genetic polymorphism, or by chemical application, can reveal the plant processes most responsive to a phytohormone. Applications of chemical inhibitors and subsequent measurements of specific phytohormones can determine whether, and which, phytohormone is affected by a molecule. In many cases, the sensitivity of biochemical testing has determined multiple pathways affected by a single inhibitor. Genetic studies are not subject to this problem, and a wealth of data about the morphological impacts of hormone biosynthetic inhibition have accumulated through the study of enzyme mutants. In this work, we sought to assess the specificity of three triazole inhibitors of cytochrome P450s by determining their abilities to recapitulate the phenotypes of single and double mutants affected in the production of brassinosteroid (BR) and gibberellin (GA) biosynthesis. The GA biosynthetic inhibitors uniconazole (UCZ) and paclobutrazol (PAC) were applied to the BR biosynthetic mutant nana plant2 (na2), and all double‐mutant phenotypes were recovered in the UCZ treatment. PAC was unable to suppress the retention of pistils in the tassels of na2 mutant plants. The BR biosynthetic inhibitor propiconazole (PCZ) suppressed tiller outgrowth in the GA biosynthetic mutant dwarf5 (d5). All treatments were additive with genetic mutants for effects on plant height. Due to additional measurements performed here but not in previous studies of the double mutants, we detected new interactions between GA and BR biosynthesis affecting the days to tassel emergence and tassel branching. These experiments, a refinement of our previous model, and a discussion of the extension of this type of work are presented.

Brassinosteroid Involvement in Arabidopsis thaliana Stomatal Opening.

Stomata within the plant epidermis regulate CO2 uptake for photosynthesis and water loss through transpiration. Stomatal opening in Arabidopsis thaliana is determined by various factors, including blue light as a signal and multiple phytohormones. Plasma membrane transporters, including H+-ATPase, K+ channels and anion channels in guard cells, mediate these processes, and the activities and expression levels of these components determine stomatal aperture. However, the regulatory mechanisms involved in these processes are not fully understood. In this study, we used infrared thermography to isolate a mutant defective in stomatal opening in response to light. The causative mutation was identified as an allele of the brassinosteroid (BR) biosynthetic mutant dwarf5. Guard cells from this mutant exhibited normal H+-ATPase activity in response to blue light, but showed reduced K+ accumulation and inward-rectifying K+ (K+in) channel activity as a consequence of decreased expression of major K+in channel genes. Consistent with these results, another BR biosynthetic mutant, det2-1, and a BR receptor mutant, bri1-6, exhibited reduced blue light-dependent stomatal opening. Furthermore, application of BR to the hydroponic culture medium completely restored stomatal opening in dwarf5 and det2-1 but not in bri1-6. However, application of BR to the epidermis of dwarf5 did not restore stomatal response. From these results, we conclude that endogenous BR acts in a long-term manner and is required in guard cells with the ability to open stomata in response to light, probably through regulation of K+in channel activity.

Overexpression of a brassinosteroid biosynthetic gene Dwarf enhances photosynthetic capacity through activation of Calvin cycle enzymes in tomato.

BackgroundGenetic manipulation of brassinosteroid (BR) biosynthesis or signaling is a promising strategy to improve crop yield and quality. However, the relationships between the BR-promoted growth and photosynthesis and the exact mechanism of BR-regulated photosynthetic capacity are not clear. Here, we generated transgenic tomato plants by overexpressing Dwarf, a BR biosynthetic gene that encodes the CYP85A1, and compared the photosynthetic capacity with the BR biosynthetic mutant dim and wild type.ResultsOverexpression of Dwarf promoted net photosynthetic rate (PN), whereas BR deficiency in dim led to a significant inhibition in PN as compared with WT. The activation status of RuBisCO, and the protein content and activity of RuBisCO activase, but not the total content and transcripts of RuBisCO were closely related to the endogenous BR levels in different genotypes. However, endogenous BR positively regulated the expression and activity of fructose-1,6-bisphosphatase. Dwarf overexpression enhanced the activity of dehydroascorbate reductase and glutathione reductase, leading to a reduced redox status, whereas BR deficiency had the contrasting effects. In addition, BR induced a reduction of 2-cystein peroxiredoxin without altering the protein content.ConclusionsBR plays a role in the regulation of photosynthesis. BR can increase the photosynthetic capacity by inducing a reduced redox status that maintains the activation states of Calvin cycle enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0715-6) contains supplementary material, which is available to authorized users.

Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA3 co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

Involvement of brassinosteroid signals in the floral-induction network of Arabidopsis

The transition to flowering is known to be regulated by numerous interacting endogenous and environmental cues, of which brassinosteroids (BRs), a group of polyhydroxylated steroid phytohormones, appear to be linked to the regulation of flowering time. In Arabidopsis, BR biosynthetic det2 mutants exhibited delayed flowering time by at least 10 d compared with the wild type. The levels of endogenous BRs in det2 were below 10% of the wild type. The timing of flowering was also delayed in the BR biosynthetic dwf4 and cpd mutants and in the BR-insensitive bri1 mutants. Because brassinolide (BL) and different BL precursors were over-accumulated in BR biosynthetic mutants and BR-insensitive bri1 mutants, this showed that alterations in the endogenous BL content and the level of different BL precursors affect flowering time in Arabidopsis. The late-flowering phenotypes of bri1 also showed that components of the BR signal transduction pathway affect flowering time. So far, reports on a connection between BRs and flowering time are limited. This review summarizes recent advances regarding the action of BRs in the transition to flowering.

Involvement of C-22-Hydroxylated Brassinosteroids in Auxin-Induced Lamina Joint Bending in Rice

The rice lamina joint is ideal material for investigating the activity of brassinosteroids (BRs) and auxin because of its high sensitivity to these compounds. Using a series of rice BR biosynthetic and receptor mutants, we conducted lamina joint tests to elucidate the mechanism of cross-talk between BR and auxin signaling in lamina joint bending. In BR biosynthetic mutants d2 and brd1, which are defective in C-23 hydroxylase and C-6 oxidase, respectively, the lamina joint response to auxin was significantly higher than that of wild-type plants. The other BR-biosynthetic mutants, brd2, osdwarf4 and d11, which are defective in C-22-hydroxylated BRs, showed less or no response to auxin. These results suggest that C-22-hydroxylated BRs are involved in auxin-induced lamina joint bending. The results were supported by the observation that inhibition of the hyper-response to auxin in d2 was reduced by treatment with brassinazole, which inhibits the function of DWARF4, the C-22 hydroxylase. In d61, which is defective in OsBRI1, a possible BR receptor in rice, the bending angle of the lamina joint in response to auxin and C-22-hydroxylated 6-deoxoBRs was nearly the same as that in wild-type plants. This implies that C-22-hydroxylated BRs function in auxin signaling independently of OsBRI1. From these observations, we propose that C-22-hydroxylated BRs participate in auxin signaling via a novel OsBRI1-independent signaling pathway.

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase.

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.

A role for brassinosteroids in germination in Arabidopsis.

This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.

BRInging light to hormone receptor activation

Brassinosteroid (BR), a plant hormone of growing interest and importance, is involved in a range of developmental processes, such as stem elongation, root growth and light signaling. The model organism, Arabidopsis, has been useful in dissecting the role of BR in plant development and photomorphogenesis. Mutant plants that are defective in BR biosynthesis develop in the dark as if they were grown in the light. One interesting mutant, bri1, resembles BR biosynthetic mutants but is insensitive to exogenous BR. The BRI gene encodes a leucine-rich repeat receptor-like kinase (LRR-RLK), the hypothesized BR receptor, the LRR being extracellular. There are >100 LRR-RLK-class genes in Arabidopsis, and so understanding how these potential receptors function is of fundamental importance.Now, Zuhua He and colleagues1xPerception of Brassinosteroids by the extracellular domain of the receptor kinase BRI1. He, Z. et al. Science. 2000; 288: 2360–2363Crossref | PubMed | Scopus (272)See all References1 report important insight into the mechanism of receptor activation in plants. The group constructed a chimeric receptor and uncoupled two domains that are necessary for specific signaling. The XA21 LRR-RLK from rice confers bacterial resistance, and resembles BRI1, the LRR and RLK domains flanking a transmembranal domain. He et al. demonstrated that a chimeric BRI1 LRR-Xa21 RLK gene activated defense responses in rice upon treatment with BR. The defense-response markers included cell death, oxidative burst and genes upregulated in disease response. One defense-response gene was induced in a BR-dose-dependent manner. Both functional BRI1 LRR and Xa21 kinase domains were necessary for the activation, as missense mutants in either domain abolished activation. The results suggest that BR binds specifically to the BRI1 LRR and activates the kinase domain, which then signals downstream responses.This study gives a clearer understanding of the primary events of BR signaling, but also paves the way for a more general mechanism of LRR-RLK activation in plants. Research suggests a model where BR interacts with the LRR domain, activating the RLK, which then transduces the signal inside the plant cell. This opens a new path of receptor research in plants, with many questions remaining. Does BR binding activate kinase activity? What is the topography of the Bri1 protein? What are downstream components of the signaling pathway? He et al. have devised a powerful approach to dissect the molecular mechanism of receptors in plants and their research points to exciting directions for the future.