BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

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.     

Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination

Seed germination is regulated by many signals. We investigated the possible involvement of a heterotrimeric G protein complex in this signal regulation. Seeds that carry a protein null mutation in the gene encoding the alpha subunit of the G protein in Arabidopsis (GPA1) are 100-fold less responsive to gibberellic acid (GA), have increased sensitivity to high levels of Glc, and have a near-wild-type germination response to abscisic acid and ethylene, indicating that GPA1 does not directly couple these signals in germination control. Seeds ectopically expressing GPA1 are at least a million-fold more responsive to GA, yet still require GA for germination. We conclude that the GPA1 indirectly operates on the GA pathway to control germination by potentiation. We propose that this potentiation is directly mediated by brassinosteroids (BR) because the BR response and synthesis mutants, bri1-5 and det2-1, respectively, share the same GA sensitivity as gpa1 seeds. Furthermore, gpa1 seeds are completely insensitive to brassinolide rescue of germination when the level of GA in seeds is reduced. A lack of BR responsiveness is also apparent in gpa1 roots and hypocotyls suggesting that BR signal transduction is likely coupled by a heterotrimeric G protein at various points in plant development.     

Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling

The Arabidopsis thaliana somatic embryogenesis receptor kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs-SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1-suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.     

Brassinosteroids regulate pectin methylesterase activity and AtPME41 expression in Arabidopsis under chilling stress

Pectin methylesterases (PMEs) are important cell wall enzymes that may play important roles in plant chilling/freezing tolerance. We investigated the possible roles of brassinosteroids (BRs) in regulation of PMEs under chilling stress. Chilling stress or 24-epibrassinolide (eBL) treatments induced significant increases in PME activity in wild type (Col-0) seedlings of Arabidopsis. The chilling-stress-induced increases in PME activity were also found in bzr1-D mutant, a BZR1 stabilized mutant with a constitutively active BR signaling pathway, but not in bri1-116, a BR insensitive null allele of the BR receptor BRI1. The results suggest that the regulation of PME activity in Arabidopsis under chilling stress depends on the BR signaling pathway. Furthermore, we showed that the effect of chilling stress on PME activity was impaired in pme41, a knockout mutant of AtPME41. Semi-quantitative RT-PCR results showed that expression of AtPME41 was induced by chilling stress in wild type plants but not in the bri1-116 mutant. The expression of AtPME41 increased in bzr1-D and eBL treated wild type seedlings, but decreased in bri1-116 seedlings. Furthermore, ion leakage induced by low temperature were dramatically increased in both bri1-116 and pme41, while lipid peroxidation was increased in bri1-116 only. The results suggest that BRs may modulate total PME activity in Arabidopsis under chilling stress by regulating AtPME41 expression. Regulation of PME activity may serve as one of the mechanisms that BR participates in chilling tolerance of plants.     

Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering

A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. In Arabidopsis thaliana, distinct genetic pathways regulate the timing of this transition. We report here that brassinosteroid (BR) signaling establishes an unexpected and previously unidentified genetic pathway in the floral-regulating network. We isolated two alleles of brassinosteroid-insensitive 1 (bri1) as enhancers of the late-flowering autonomous-pathway mutant luminidependens (ld). bri1 was found to predominantly function as a flowering-time enhancer. Further analyses of double mutants between bri1 and known flowering-time mutants revealed that bri1 also enhances the phenotype of the autonomous mutant fca and of the dominant FRI line. Moreover, all of these double mutants exhibited elevated expression of the potent floral repressor FLOWERING LOCUS C (FLC). This molecular response could be efficiently suppressed by vernalization, leading to accelerated flowering. Additionally, specific reduction of the expression of FLC via RNA interference accelerated flowering in bri1 ld double mutants. Importantly, combining the BR-deficient mutant cpd with ld also resulted in delayed flowering and led to elevated FLC expression. Finally, we found increased histone H3 acetylation at FLC chromatin in bri1 ld mutants, as compared with ld single mutants. In conclusion, we propose that BR signaling acts to repress FLC expression, particularly in genetic situations, with, for example, dominant FRI alleles or autonomous-pathway mutants, in which FLC is activated.     

Allele-specific suppression of a defective brassinosteroid receptor reveals a physiological role of UGGT in ER quality control

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a presumed folding sensor of protein quality control in the endoplasmic reticulum (ER). Previous biochemical studies with nonphysiological substrates revealed that UGGT can glucosylate nonnative glycoproteins by recognizing subtle folding defects; however, its physiological function remains undefined. Here, we show that mutations in the Arabidopsis EBS1 gene suppressed the growth defects of a brassinosteroid (BR) receptor mutant, bri1-9, in an allele-specific manner by restoring its BR sensitivity. Using a map-based cloning strategy, we discovered that EBS1 encodes the Arabidopsis homolog of UGGT. We demonstrated that bri1-9 is retained in the ER through interactions with several ER chaperones and that ebs1 mutations significantly reduce the stringency of the retention-based ER quality control, allowing export of the structurally imperfect yet biochemically competent bri1-9 to the cell surface for BR perception. Thus, our discovery provides genetic support for a physiological role of UGGT in high-fidelity ER quality control.     

Suppressor of <Emphasis Type="Italic">bri1-120</Emphasis> mutant allele revealed interrelated and independent actions of brassinosteroid and light signaling

Brassinosteroids (BRs) are plant hormones that affect diverse aspects of plant development. Various BR-biosynthetic or BR-signaling mutants contribute to BR functions and signaling events in many plant species. The BR receptor brassinosteroid-Insensitive 1 (BRI1) plays critical roles in BR signaling. We previously identified a weak bri1 mutant allele, bri1-120, that has a mutation site in the extracellular domain of BRI1. Here, genetic suppressor screening revealed that a PHYB gene mutation led to suppression of ethyl methanesulfonate (EMS)-mutagenized bri1-120. The morphology of bri1-120phyB-1 indicated that compact and rounded phenotypes of bri1-120 were suppressed. However, BR sensitivity of the bri1-120phyB-1 was only recovered in hypocotyl elongation, and overexpression of PHYB in bri1-120 did not enhance bri1-120 phenotypes. To further investigate the relationship between BR and light signalings, we examined the seed germination pattern and hypocotyl growth of bri1-120phyB-1 as compared to that of each single mutant under various light conditions. Seed germination in bri1-120phyB-1 was higher than in both the single mutants. Hypocotyl length in bri1-120phyB-1 was intermediate between that of bri1-120 and phyB-1, whereas sensitivity to red light in bri1-120phyB-1 remained the same as in phyB-1. These results suggest that BR and light signalings affect diverse cellular responses both together and independently, depending on the specific cellular processes.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

Loss-of-function mutations in the Arabidopsis heterotrimeric G-protein alpha subunit enhance the developmental defects of brassinosteroid signaling and biosynthesis mutants

Loss-of-function alleles of the sole heterotrimeric G-protein alpha subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development. Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response. Here we provide genetic evidence that loss-of-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis. gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively. By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants. Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants. Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots. Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner.     

BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling

The Arabidopsis BAK1 (BRI1 Associated receptor Kinase 1) was identified by a yeast two-hybrid screen as a specific interactor for BRI1, a critical component of a membrane brassinosteroid (BR) receptor. In yeast, BAK1/BRI1 interaction activates their kinase activities through transphosphorylation. BAK1 and BRI1 share similar gene expression and subcellular localization patterns and physically associate with each other in plants. Overexpression of the BAK1 gene leads to a phenotype reminiscent of BRI1-overexpression transgenic plants and rescues a weak bri1 mutant. In contrast, a bak1 knockout mutation gives rise to a weak bri1-like phenotype and enhances a weak bri1 mutation. We propose that BAK1 and BRI1 function together to mediate plant steroid signaling.     

Proteomic study identifies proteins involved in brassinosteroid regulation of rice growth

Brassinosteroids (BRs) are essential hormones for growth and development of plant. In rice, BRs regulate multiple developmental processes and affect many important traits such as height, leaf angle, fertility and seed filling. We identified brassinosteroid-regulated proteins in rice using proteomic approaches and performed functional analysis of some BR-regulated proteins by overexpression experiments. Using two-dimensional difference gel electrophoresis (2-D DIGE) followed by protein identification by mass spectrometry, we compared proteomic differences in the shoots and roots of the BR-insensitive mutant d61-4 and BR-deficient mutant brd1-3. We identified a large number of proteins differentially expressed in the mutants compared with wild type control. These include a glycine-rich RNA-binding protein (OsGRP1) and a DREPP2 protein, which showed reduced levels in the BR mutants. Overexpression of these two proteins partially suppressed the dwarf phenotype of the Arabidopsis BR-insensitive mutant bri1-5. In contrast to the reduced protein level, the RNA level of OsGRP1 was not significantly affected in the BR mutants or by BR treatment, suggesting BR regulation of OsGRP1 at the posttranslational level. This study identifies many BR-regulated proteins and demonstrates that OsGRP1 functions downstream in the BR signal transduction pathway to promote cell expansion.     

Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels.     

Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.     

The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth

Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.