Intragenic Suppression of a Trafficking-Defective Brassinosteroid Receptor Mutant in Arabidopsis

Abstract

The cell surface receptor kinase BRASSINOSTEROID-INSENSITIVE-1 (BRI1) is the major receptor for steroid hormones in Arabidopsis. Plants homozygous for loss-of-function mutations in BRI1 display a reduction in the size of vegetative organs, resulting in dwarfism. The recessive bri1-5 mutation produces receptors that do not accumulate to wild-type levels and are retained mainly in the endoplasmic reticulum. We have isolated a dominant suppressor of the dwarf phenotype of bri1-5 plants. We show that this suppression is caused by a second-site mutation in BRI1, bri1-5R1. The bri1-5R1 mutation partially rescues the phenotypes of bri1-5 in many tissues and enhances bri1-5 phenotypes above wild-type levels in several other tissues. We demonstrate that the phenotypes of bri1-5R1 plants are due to both increased cell expansion and increased cell division. To test the mechanism of bri1-5 suppression, we assessed whether the phenotypic suppression in bri1-5R1 was dependent on ligand availability and the integrity of the signaling pathway. Our results indicate that the suppression of the dwarf phenotypes associated with bri1-5R1 requires both BR biosynthesis and the receptor kinase BRI1-ASSOCIATED KINASE-1 (BAK1). Finally, we show that bri1-5R1 partially restores the accumulation and plasma membrane localization of BRI1. Collectively, our results point toward a model in which bri1-R1 compensates for the protein-folding abnormalities caused by bri1-5, restoring accumulation of the receptor and its delivery to the cell surface.

STEROIDS are important small-molecule hormones throughout the plant and animal kingdoms. The structure of the first plant brassinosteroid (BR) was determined almost 30 years ago (Grove et al. 1979), and the first major bioactivities associated with BR application were the stimulation of cell elongation and cell division (Mandava 1988). BRs are widely distributed in the plant kingdom (Bishop 2003). BRs gained attention as a major class of plant hormones after the analysis of Arabidopsis mutants deficient in BR biosynthesis (reviewed in Clouse and Feldmann 1999). These mutants showed major developmental defects including dwarfism, dark-green leaves, male sterility, and delays in flowering and senescence. The dwarfism, which was mainly due to shorter organs such as leaves and stems, could be partly reversed by supplementing mutant plants with end products of BR biosynthesis (Szekeres et al. 1996).

While steroids serve as signaling molecules in both plants and animals, the mechanisms through which they are perceived appear to be distinct. The primary animal steroid receptors are transcription factors, such as the estrogen receptor, whose ligand binding triggers DNA association and the induction or repression of specific genes (Rosenfeld and Glass 2001). The nuclear hormone receptors found in animals are likely not involved in plant steroid perception, as this family was not identifiable in the Arabidopsis genome (Arabidopsis Genome Initiative 2000). Rather, analysis of steroid responses in plants has revealed that the major mechanism of steroid perception is via plasma membrane-spanning receptors (Li and Chory 1997; Li et al. 2002; Nam and Li 2002).

The cellular activities of BRs are mediated by their interaction with a specific cell-surface receptor that belongs to the leucine-rich repeat–receptor-like kinase (LRR–RLK) family, a group of structurally related plant receptors (Dievart and Clark 2003). BRASSINOSTEROID INSENSITIVE-1 (BRI1) was identified through genetic screens for Arabidopsis mutants displaying a dwarf stature that could not be rescued by exogenous BR treatment (Clouse et al. 1996; Li and Chory 1997). BRI1 contains an extracellular domain (ECD) composed of an N-terminal signal sequence followed by 25 LRRs (Figure 1C; Li and Chory 1997). LRRs form a highly reiterative structure shaped like a horseshoe, providing surfaces for interaction with ligands or other molecules (Bell et al. 2003). BRI1 co-immunoprecipitates with active BRs, and photoaffinity experiments demonstrate that the binding site for BRs is an atypical LRR, LRR22, and a 70-amino-acid “island” found between LRRs 21 and 22 (Kinoshita et al. 2005). The ECD of BRI1 is followed by a single transmembrane pass and a cytoplasmic Ser/Thr kinase domain (Figure 1B), the latter of which is maintained in its basal resting state through the activities of both its C-terminal tail and a membrane-associated protein, BRI1 Kinase Inhibitor-1 (BKI1) (Friedrichsen et al. 2000; Wang et al. 2005a,b; Wang and Chory 2006). Recently, the kinase domain of BRI1 has been shown to belong to the superfamily of dual-specificity kinases and is able to display, in addition to its Ser/Thr kinase activity, a Tyr kinase activity in planta (Oh et al. 2009). The Tyr kinase activity of BRI1 is required for full receptor function and acts in an additive fashion to the Ser/Thr kinase activity. Thus, the early activation of the pathway depends on the ordered and regulated autophosphorylation of BRI1 by both its Ser/Thr- and Tyr-kinase activity, suggesting that distinct phosphorylation states may fine-tune the receptor signaling capacity.

Figure 1.—

A comparison of bri1-5R1 phenotypes with other mutants in steroid signaling, genetic interactions with steroid-signaling mutants and a comparison of other known mutations in extracellular LRRs of RLKs. (A) Photographs of 6-week-old Ws-2, bak1-2, bri1-5R1, bri1-5, bak1-2 bri1-5R1, bak1-1 bri1-5, bin2-1, bin2-1 bri1-5R1, and bri1-4 plants. bak1-1 bri1-5 plants were described in Li et al. (2002). (B) Schematic of the domain organization of BRI1. The locations of the missense mutations bri1-5, bri1-R1, and bri1-9 and the deletion mutation bri1-4 in the extracellular domain of BRI1 are indicated by triangles. (C) Location of mutations in extracellular LRRs from Arabidopsis RLKs. The BRI1 consensus sequence is from Li and Chory (1997), the clv1 mutations (D to N, G to E) from Dievart et al. (2003), FLS2 from Gomez-Gomez and Boller (2000), elg from Whippo and Hangarter (2005), and bri1-9 from Noguchi et al. (1999). (D) Clustering of the bri1-5R1 mutation in the highly conserved N-terminal Cys capping domain. (Top) Alignment of the N-terminal Cys capping domains of AtBRI1 and bri1-5R1. (Middle) Alignment of the N-terminal Cys capping domains of BRI1 orthologs across dicots (Middle top) and monocots (Middle bottom), highlighting the residues mutated in our bri1-5R1 allele in red. Proteins compared were the following: dicots—LeBRI1 (tomato), SbBRI1 (potato), NbBRI1 (Nicotiana benthamiana, tobacco), and NtBRI1 (Nicotiana tabacum, tobacco); monocots—HvBRI1 (barley), TaBRI1 (wheat), and OsBRI1(rice). (Bottom) Alignment of the N-terminal Cys capping domains of other closely related LRR–RLKs in Arabidopsis, including BAK1, a coreceptor for BRI1. AtBRL1 and BRL3 are BRI1 paralogs and CLV1 and FLS2 are phylogenetically related to BRI1. Residues related to this study are shown in red.

In vivo cellular biology approaches showed that the BRI1 protein is localized primarily to the plasma membrane, where the perception of steroids is likely to occur (Friedrichsen et al. 2000; Kinoshita et al. 2005; Geldner et al. 2007). In addition to its plasma membrane localization, BRI1 is present in intracellular endosomal compartments (Russinova et al. 2004; Geldner et al. 2007). While the distribution of BRI1 in the endosomal pools is not affected either by exogenous treatment with BRs or by endogenous BR depletion, several lines of evidence indicate that BRI1 has the ability to signal from these intracellular compartments. This intracellular localization of the receptor is reminiscent of TGF-β signaling in animals where endocytosis of the TGF-β receptor is necessary for signal transmission (Raikhel and Hicks 2007). Taken together, the BR insensitivity of bri1 mutants, the plasma membrane localization of the receptor, and the ability of BRs to bind to the extracellar LRRs demonstrate that BRs are sensed by the LRR module of BRI1 at the cell surface (Belkhadir and Chory 2006). However, it remains unclear how the binding of BRs regulates the phosphorylation state of the receptor.

Initially, the only types of mutants identified in screens for BR-insensitive plants were loss-of-function mutations in BRI1. This suggests that additional positive regulators in this pathway are lethal or redundant. Both second-site suppressor screens and biochemical methods were used to bypass these genetic limitations to identify other BR-signaling components. BRI1-ASSOCIATED KINASE-1 (BAK1) was one locus identified through alternate approaches (Li et al. 2002; Nam and Li 2002). A yeast two-hybrid screen using the BRI1 kinase domain as a prey identified BAK1 as a BRI1 interactor (Nam and Li 2002). In addition, BAK1 was also found to be a dose-responsive suppressor of the weak BRI1 allele bri1-5 in an activation-tagging screen (Li et al. 2002). The weak dwarf phenotype associated with loss-of-function bak1 alleles is likely due to the overlapping functions of its paralogs, a small subfamily of five LRR–RLKs called the SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) (Hecht et al. 2001; Albrecht et al. 2008). A striking structural difference between BRI1 and BAK1 lies in the number of LRRs that they possess. BAK1 has only five extracellular LRR repeats and no obvious platform for a BR-binding domain (Belkhadir and Chory 2006). Furthermore, the binding of BRs to BRI1 has been shown to be BAK1 independent. The binding of BRs to preformed BRI1 oligomers is immediately followed by the activation of the kinase domain, leading to autophosphorylation of the receptor and subsequent phosphorylation of BAK1 after the displacement of BKI1 from the plasma membrane (Wang et al. 2005a,b; Wang and Chory 2006).

Currently, the only known downstream factors of BRI1–BAK1 complexes are the BRASSINOSTEROID SIGNALING KINASES (BSKs) (Tang et al. 2008). Recent models propose that, in the presence of BRs, the phosphorylation of BSK1 by BRI1 promotes the binding of BSK1 to the phosphatase BRI1 Suppressor-1 (BSU1). Consequently, BSU1 is able to inactivate the BRASSINOSTEROID INSENSITIVE-2 (BIN2) kinase (Li and Nam 2002; Vert and Chory 2006) by targeting BIN2 for dephosphorylation (Kim et al. 2009). The BSU1-dependent inactivation of BIN2 allows the transcription factors BRI1 EMS SUPPRESSOR-1 (BES1) and BRASSINAZOLE RESISTANT-1 (BZR1) to activate growth responses (Vert and Chory 2006). BZR1 plays a critical role in feedback signaling by repressing BR biosynthetic genes transcriptionally in response to BR perception (He et al. 2005). Conversely, in the absence of BRs, BIN2 phosphorylates BES1and BZR1, which targets them for degradation (Yin et al. 2002; He et al. 2005).

While there is strong evidence that the kinase domains of BRI1 and BAK1 physically interact and phosphorylate each other, the interactions between their ECDs are less clear. Many LRR–RLKs contain two pairs of cysteines in their ECDs: one at their amino terminus and one near the transmembrane domain. These have been proposed to function in the homodimerization or heterodimerization of RLKs (Dievart and Clark 2003), although there is no direct evidence for this yet. Recent findings favor a model in which the disulphide-bonded cysteine caps flanking both ends of an LRR platform would function by protecting the exposed edges of the hydrophobic core formed by the canonical LRRs and by helping the LRRs to fold along an N-terminal polarized pathway in which the N-terminal cysteine-rich capping domain is used as a nucleation point for proper folding to proceed (Courtemanche and Barrick 2008; Truhlar and Komives 2008).

The relative lack of missense mutations in the ECD of BRI1 as compared to its kinase domain suggests that there are fewer key individual amino acids in the ECD of BRI1 compared to the cytosolic domain. Alternatively, the LRR platform, because of its versatility, may tolerate amino acid substitution better than the kinase domain. The bri1-9 mutation, which confers weak insensitivity to BRs, is located in the 22nd LRR and may interfere with ligand binding (Noguchi et al. 1999; Kinoshita et al. 2005). However, analysis of an allele-specific suppressor of bri1-9 revealed that BRI1-9 is a functional receptor, but is retained in the Endoplasmic Reticulum (ER) by the collective action of a plant-specific calreticulin (CRT3) (Jin et al. 2009) and an α1,6 mannosyltransferase (Hong et al. 2009) for targeted degradation by a proteasome-mediated process (Jin et al. 2007; Hong et al. 2008, 2009).

To identify essential regions of BRI1, we have performed a suppressor screen with bri1-5, a hypomorphic mutation in BRI1 that changes one of the paired cysteines in the amino-terminus of BRI1 to a tyrosine. bri1-5, like bri1-9, encodes a receptor variant that does not accumulate to wild-type levels and is retained in the ER, likely by similar processes (Jin et al. 2007; Hong et al. 2008, 2009). In this study, we show that one of our suppressor mutants, bri1-5R1, is an intragenic revertant of bri1-5. bri1-5R1 is a mutation in the first LRR of BRI1, only 20 amino acids from the bri1-5 mutation. Our analysis of bri1-5 and bri1-5R1 phenotypes reveals that bri1-5R1 plants display many intermediate phenotypes compared to wild-type and bri1-5 plants. However, bri1-5R1 also confers several gain-of-function phenotypes, including increased pedicel length. We demonstrate that the bri1-5R1 phenotypes require BR biosynthesis and the coreceptor BAK1. Our results suggest that bri1-5R1 acts primarily by allowing the trafficking-defective receptor to exit the ER and to reach the cell surface where it can perceive its ligand.

MATERIALS AND METHODS

Isolation, mapping, and sequencing of bri1-5R1:

Mutagenesis:

Seeds homozygous for bri1-5 in the Wassilewskija-2 (Ws-2) accession were mutagenized with EMS as described previously (Zhao et al. 2002)

Mapping:

One revertant strain (4-8) was intermediate in height between bri1-5 and Ws-2 plants. The strain 4-8 was outcrossed five times to segregate out additional mutations and crossed to bri1-9 plants in the Columbia accession for RFLP mapping. DNA was isolated from F₂ plants resembling 4-8 and tested using nga 1107, an RFLP marker closely linked to BRI1 (Bell and Ecker 1994).

Complementation test with bri1-4:

To test whether 4-8 was an allele of BRI1, we crossed 4-8 plants to plants heterozygous for bri1-4, a null allele of BRI1 (Noguchi et al. 1999). Half of the F₁ plants were wild type in appearance (bri1-5 4-8/+) and half showed the 4-8 phenotype (bri1-5 4-8/bri1-4). The genotypes of the two phenotypic classes were confirmed with PCR (see below).

Sequencing of bri1-5R1:

DNA sequencing, including PCR fragments and primers, was performed as described by (Noguchi et al. 1999). The bri1-5R1 mutation generates a new EcoRI site, which was used to distinguish between mutant and wild-type alleles.

Phenotypic comparison of bri1-5 and bri1-5R1 to wild-type plants:

Growth of plants:

Approximately five seeds were planted in round pots (5 cm in diameter) with soil (4 parts Sunshine mix 3, Sungro Horticulture:1 part vermiculite, CAS# 1318-00-9, Therm-O-Rock West) presoaked in water. Flats containing the pots were covered in plastic wrap and cold treated for 3–4 days before transfer to a Conviron MTR30 growth chamber [16 hr of light and 8 hr of dark at 22°, with 75% humidity, and a light intensity of ∼120 μE/m² from cool-white fluorescent tube lamps (Philips F96/CW/VHO) supplemented by incandescent bulbs]. Plastic wrap was removed after the seedlings were established (after 3–5 days), and one representative seedling was allowed to grow per pot. The pots were sub-irrigated with Hoagland's nutrient solution as necessary. When the plants were 5 weeks of age, some of the morphological traits listed in Table 1 were measured. Other traits were measured after the plants had ceased growth. Plant height was measured to the nearest millimeter, and the length of siliques, the distance between siliques on the main inflorescence, and the length and width of leaves were measured to the nearest half-millimeter using a ruler. Differences among genotypes were tested using analysis of variance (ANOVA).

TABLE 1.

Morphometric analysis of Ws-2, bri1-5, bri1-5R1, bak1-2, and bri1-5R1bak1-2 mutants

Attribute	Ws-2	bri1-5	bri1-5R1	bak1-2	bri1-5R1bak1-2
Heighta (in cm, 7 weeks old, n > 18)	34.9 ± 4.64	9.88 ± 1.32	21.9 ± 3.18	36.2 ± 5.38	9.79 ± 1.85
Distance between siliques on main inflorescencea (in cm, n > 50)	0.89 ± 0.4	0.26 ± 0.18	0.60 ± 0.49	1.07 ± 0.69	0.31 ± 0.26
Length of pedicelsb (in cm, n > 50)	1.21 ± 0.25	1.16 ± 0.17	1.94 ± 0.23	0.91 ± 0.15	1.48 ± 0.14
Length of siliques (in mm, n = 50)	11.9 ± 0.83	7.08 ± 0.43	10.2 ± 1.4
No. of rosette leaves (n = 20)	5.85 ± 1.0	7.65 ± 1.17	8.35 ± 0.88
Width of rosette leaves (in cm, n > 115)	0.86 ± 0.35	1.0 ± 0.35	0.93 ± 0.29
Length of rosette leaves (in cm, n > 115)	2.6 ± 0.8	1.8 ± 0.6	2.6 ± 0.8
No. of cauline leaves (n = 20)	1.7 ± 0.8	2.2 ± 1.03	3.3 ± 0.74
Width of cauline leaves (in cm, n > 39)	0.49 ± 0.3	0.45 ± 0.2	0.43 ± 0.14
Length of cauline leaves (in cm, n > 39)	1.9 ± 0.44	1.84 ± 0.44	2.2 ± 0.51
No. of seeds (n = 50 siliques)	50.0 ± 4.8	30.3 ± 3.6	40.4 ± 5.8
No. of floral organs (n = 200):
Sepals**	4.02 ± 0.12	4.02 ± 0.14	4.22 ± 0.44
Petals**	4.02 ± 0.14	4.02 ± 0.14	4.13 ± 0.40
Anthers*	5.88 ± 0.39	5.96 ± 0.23	6.04 ± 0.34
Valves***	2.01 ± 0.08	2.22 ± 0.47	2.21 ± 0.40

Each value represents the mean ± SD. *P < 0 .05, one-way ANOVA for all three genotypes; **P < 0.5, one-way ANOVA that bri1-5R1 is different from Ws-2 and bri1-5; ***P < 0.05, one-way ANOVA that bri1-5R1 and bri1-5 are different from Ws-2.

^aP < 0.05, one-way ANOVA that Ws-2 and bak1-2 are not different, bri1-5 and bri1-5R1 bak1-2 are not different, and bri1-5R1 is different from all others.

^bP < 0.05, one-way ANOVA that Ws-2 and bri1-5 are not different from each other, but all the rest are different.

Seedling responses to BRs and other chemicals:

Plate assays were performed using surface-sterilized seeds, which were soaked in 70% ethanol and 0.1% Triton X-100 for 10 min and then rinsed twice with 95% ethanol. Seedlings were grown on 1% agar plates with 0.5× Murashige and Skoog (MS) media and 0.5% 2-(N-morpholino)ethane sulfonic acid, pH 5.7. Plates were sealed with Micropore surgical tape (3M), stratified in the dark at 4° for 72 hr and grown vertically at 22°, with 16 hr light/8 hr dark, under cool-white fluorescent tubes (Phillips Alto F40CW/RS/EW) with an average output of 90 μE/m². For root-length assays, plates were scanned with a HP ScanJet5370C and roots were measured using National Institutes of Health (NIH) ImageJ (NCBI). BR root-growth inhibition for Ws-2, bri1-5, and bri1-5R1 was assayed as in Clouse et al. (1996) on plates containing 0, 5 × 10⁻⁵, 1 × 10⁻⁴, 5 × 10⁻⁴, 1 × 10⁻³, 5 × 10⁻³, 1 × 10⁻², 5 × 10⁻², 0.1, 0.5, and 1 μm epi-brassinolide (epiBL) (Sigma E1641), measuring after 7 days of growth; n > 20 for each data point ± 1 SD. Seedling responses to brassinazole and kifunensine (Kif) were assayed as described (Wang et al. 2002; Jin et al. 2007).

Histological analysis:

Stem and pedicel tissue was collected from 3-week-old plants. Stem samples were collected from between the first and second siliques, and pedicels were collected from the first and second siliques. The samples were embedded in 1% agarose and fixed in 2% gluteraldehyde in 1× phosphate-buffered saline at room temperature for 3 hr. The samples were then dehydrated using an ethanol series of 10, 20, 30, 40, 50, 60, 70, 80, and 90% ethanol for 15 min, followed by three washes in 100% ethanol. After dehydration, LR White embedding resin (Electron Microscopy Sciences, Hatfield, PA) was added in 1/4 volume to 100% ethanol every 4 hr, followed by three changes of 100% LR White. After infiltration, the samples were embedded in gelatin capsules or BEEM narrow-neck molds (Electron Microscopy Sciences) and baked at 61° overnight. Sections ∼2- to 7-μm thick were created using a Sorvall MT2B Ultramicrotome, stained with 0.1% methylene blue, and visualized under ×5 magnification on a Zeiss Axioplan compound microscope equipped with a QImaging Micro Publisher 3.3 RTV camera. Measurements and counts from 10 plants were performed using NIH ImageJ Software (NCBI). All graphs were prepared using Microsoft Excel, and error bars represent 1 SD above and below the mean. Statistical differences in average cell lengths were analyzed using ANOVA.

Analysis of BR levels:

Aerial parts of soil-grown plants (5 weeks old, ∼30 g fresh weight; see Phenotypic comparison of bri1-5 and bri1-5R1 to wild-type plants, above, for growing conditions) were harvested and lyophilized. The tissues were extracted twice with 500 ml of methyl alcohol. Deuterium-labeled internal standards were added to the extracts. Purification and quantification of sterols and BRs were carried out according to the method described by (Fujioka et al. 2002).

Protein analysis and EndoH treatment:

Two-week-old seedlings were first ground in liquid nitrogen with a mortar and pestle. This material was then homogenized by alternate rounds of Polytron (Kinematica) in 2 ml of sterile buffer (20 mm Tris–HCl, pH 8.0, 0.33 m sucrose, 10 mm EDTA, 5 mm dithiothreitol, and plant protease inhibitor cocktail (Roche)/1 g of tissue). Debris was removed by centrifugation at 2000 × g for 10 min at 4°. The supernatant of the 2000 × g spin was then centrifuged at 20,000 × g for 45 min. For detection of BRI1, the pellet from the 20,000 × g spin was extracted in 2.5× Laemli buffer. For immunodetection, samples were electrophoresed on 6% SDS–polyacrylamide gels. Western blots were performed using standard methods and detected with ECL+ (Amersham). Proteins were subjected to deglycosylation assays by treatment with endoglycosidase H by following the manufacturers' instructions.

Genetic interactions with BR biosynthetic and signaling mutants:

bri1-5R1 plants were crossed with plants containing mutations in bin2 (dwf12-1D; Choe et al. 2002), dwf4-1 (Choe et al. 1998), cpd-5 (Choe 2004), and bak1-2 (Li et al. 2002) to assay for genetic interactions. The F₁ generation was screened via PCR for plants that were doubly heterozygous to ensure that the cross was successful, and these plants were then selfed to obtain double homozygous mutants. For dwf4-1 and cpd-5, which are unlinked to bri1-5R1, strains homozygous for bri1-5R1 were identified, and phenotypes of the progeny that were segregating for dwf4-1 or cpd-5 were monitored. Since BAK1 and BIN2 are linked to BRI1, the progeny from these crosses were planted and then screened for F₂ plants that were heterozygous for one mutation and homozygous for the other using PCR (see below). These F₂ plants were self-crossed, and progeny seeds were collected and planted to obtain double homozygous mutants. Double mutants were confirmed by PCR and digestion of products. For identifying insertion mutants, PCR was performed using two primers that amplify the genomic DNA and a third border primer that amplifies the T-DNA/plant DNA junction. For point mutants, cleaved amplified polymorphic sequences (CAPS) or derived CAPS (dCAPS) primers were made using the dCAPS program (Neff et al. 2002), and PCR-amplified DNA was cut with an enzyme that distinguishes mutants from wild type. The progeny of the double homozygous mutants were then characterized phenotypically and photographed. All mutations except cpd-5 were originally isolated in the Ws-2 accession. Our results with cpd-5 and dwf4-1 were very similar, indicating that the Enkheim accession background of cpd-5 did not influence these genetic interactions.

PCR primers and CAPS enzymes:

The following primers and enzymes were used in our study:

• bak1-2 primers: sense—5′-CTTCCAAGTCTTAATCTGATGGGCCTTTA-3′; anti-sense—5′-GCAGGTGATGGCGGTGTAGGAGAGATAGG-3′; JL-202 (left border)—5′-CATTTTATAATAACGCTGCGGACATCTAC-3′;

• bri1-4 CAPS primers: sense—5′-TGTTTCTCTCAAACTCACACATCAA-3′; anti-sense—5′-CGAATTGGTTACTAGAGATGTTCAA-3′; bri1-4 contains a 10-bp deletion, 458 bp downstream of ATG, and results in the loss of a BsmAI site;

• bri1-5 dCAPS primers: sense—5′-CCGTGTACTTTCGATGGCGTTACCT-3′; anti-sense—5′-CAAGCTGGTTAAAGAAGCAGAGCAC-3′; bri1-5 contains the mutation G206A (C69Y). The underlined base in the sense primer is an introduced dCAPS mismatch causing the gain of a PstI site in wild type, but not in bri1-5.

• bri1-5R1 CAPS primers: sense—5′-TAATCAGAAGAAGAGGTAAC-3′; antisense—5′-TTCCCGGAGATGTCAAGATG-3′; bri1-5R1 contains the mutation G260A (G87E) and results in the gain of an EcoRI site;

• bin2-1 CAPS primers: sense—5′-TCTTGGTCAGGTAAACAATTCTTTCAGT-3′; antisense—5′-AAAGAAACTGAAACAAGAACACATGCAA-3′; bin2-1 contains the mutation G989A (E264K) and results in the loss of an MboII site.

RESULTS

Identification of an intragenic suppressor mutation in the BRI1 gene:

To understand how the BRI1 receptor kinase connects BR perception to cellular responses, we carried out a suppressor screen by looking for revertants of the dwarf phenotype of plants homozygous for the weak bri1-5 mutation. In addition to identifying five extragenic suppressors in BES1 (Zhao et al. 2002), we also found a suppressor, initially named 4-8, with a unique phenotype. This phenotype includes a semi-dwarf stature intermediate between that of bri1-5 plants and that of the wild type, rounded leaves, and long pedicels (Figure 1A and Table 1). Plants derived from seeds of the original 4-8 strain were outcrossed to plants of the Wassilewskija-2 (Ws-2) accession. The F₁ progeny from these crosses were wild type in appearance, indicating that 4-8 is recessive. Plants resembling the original 4-8 plants were found in an ∼1:3 ratio (nine 4-8:24 wild type) in the F₂ from these crosses, and no plants resembling bri1-5 were identified. This indicated that the 4-8 mutation was homozygous in the original strain and suggested that 4-8 was linked to bri1-5.

To more precisely determine the linkage of the 4-8 mutant phenotypes to bri1-5, 4-8 mutants were crossed to plants homozygous for the weak bri1-9 mutation in the Columbia accession (Jin et al. 2007). The F₁ from this cross phenotypically resembled 4-8 plants, indicating that 4-8 is dominant to the bri1-9 allele. Analysis of DNA from 40 F₂ plants with a bri1-9 phenotype identified only one recombinant chromosome for the marker nga1107, which is tightly linked to BRI1.

To test whether 4-8 was an allele of BRI1, we performed a complementation test with a null allele for BRI1. Plants homozygous for 4-8 were crossed to plants heterozygous for the bri1-4 mutation, a 10-bp deletion in the extracellular domain of BRI1 (Noguchi et al. 1999). Approximately half of the progeny from this cross are expected to be 4-8/+, with the remainder being 4-8/bri1-4. Half of the progeny were wild type in appearance, and the other half resembled 4-8, indicating that 4-8 fails to complement bri1-4 and is an allele of BRI1. PCR analysis of the 10-bp deletion in bri1-4 and of the 4-8 mutation confirmed that plants resembling 4-8 were 4-8/bri1-4.

We sequenced the BRI1 coding sequence in 4-8 and identified, in addition to the bri1-5 mutation, a mutation in the first LRR (G260A, Gly87Glu). The first LRR in BRI1 has been described as a partial LRR because it does not contain all of the residues typical of the LRR consensus for BRI1 (Li and Chory 1997). The glycine altered in bri1-5R1 is highly conserved in the LRRs of BRI1 and other prototypical RLKs (Figure 1D). This new mutation was named bri1-R1, and when present together with the original bri1-5 mutation, is called bri1-5R1; this terminology will be used here to refer to the revertant strain.

Phenotypic characterization of bri1-5R1 plants:

The bri1-5R1 mutant was selected as a revertant because of its increased size relative to bri1-5 (Figure 1A). Before further investigating the morphology of bri1-5R1 plants, we performed five backcrosses to the wild-type Ws-2 accession to remove possible background mutations. Progeny of the final backcross plants were grown with bri1-5 and Ws-2 plants under controlled conditions in which plant organ sizes were measured and compared (Table 1). In general, bri1-5R1 plants were intermediate between bri1-5 and Ws-2 plants for specific traits such as plant height, distance between internodes, silique length, and seed number per seedpod. However, bri1-5R1 plants had longer pedicels than bri1-5 or Ws-2 plants. bri1-5R1 plants had slightly more sepals and petals than both bri1-5 and Ws-2 plants and also produced more valves in the mature fruit than Ws-2 plants produced, but a similar number of valves were also generated by bri1-5 plants.

The increased size of bri1-5R1 plants compared to bri1-5 plants appeared to be due to an increase in the lengths of internodes. To test if this increase was due to increased cell expansion, cell division, or both, transverse sections of stems were made, and epidermal cell length was measured. We found that the mean stem epidermal cell length in the bri1-5R1 plants was statistically shorter than Ws (0.0654 ± 0.0231 mm vs. 0.109 ± 0.0518 mm) and longer than bri1-5 (0.0371 ± 0.0148 mm, P < 0.01, one-way ANOVA), indicating that bri1-5R1 is intermediate in cell length between Ws-2 and bri1-5; however, we observed a large variation in cell length in both Ws-2 and bri1-5R1 (Figure 2, A–C). Therefore, we performed a more detailed analysis of the distribution of cell length and found that, while the range of cell lengths observed in both Ws-2 and the mutants varied greatly, the majority of cells in bri1-5R1 were intermediate in length between bri1-5 and Ws-2 and that bri1-5R1 never produces cells as long as Ws-2 (Figure 2G). These results suggest that the size increase seen in stems from bri1-5R1 plants is caused by the reversion of the cell expansion defects of bri1-5.

Figure 2.—

Analysis of cell division and cell expansion in bri1-5 and bri1-5R1 mutants. Transverse sections of Ws-2 (A), bri1-5R1 (B), and bri1-5 (C) stems and Ws-2 (D), bri1-5R1 (E), and bri1-5 (F) pedicels stained with 0.1% methylene blue to visualize cells. Bars, 0.1 mm. Due to the variation in cell length, we analyzed the distribution of cell lengths for Ws-2, bri1-5, and bri1-5R1 stem epidermal cells (G) and pedicel epidermal cells (H). In the stems, a majority of cells in bri1-5 plants were very short, whereas in bri1-5R1 most were intermediate, and in Ws-2, the cells were more evenly distributed with more cells being longer in length, supporting the hypothesis that the height differences observed in these plants are due to cell expansion defects. In the pedicels, no significant difference was observed in the distribution between Ws-2 and bri1-5 plants, while the bri1-5R1 genotype was found to have more cells of longer length.

bri1-5R1 mutants also generated significantly longer pedicels as compared to Ws-2 (Table 1). To determine whether this phenotype is also due to an increase in cell expansion, we sectioned and measured pedicel epidermal cell length (Figure 2D, E and F). We found that, while there was no significant difference in average cell length between Ws-2 (0.0759 ± 0.0406 mm) and bri1-5 (0.0747 ± 0.0363 mm, P < 0.01 one-way ANOVA), the average cell length in bri1-5R1 pedicels was significantly higher than Ws-2 and bri1-5 (0.0912 ± 0.0433 mm, P < 0.001 one-way ANOVA). However, when the distribution of cell lengths was examined for pedicel cells, we found that it was only slightly skewed toward having a greater number of more expanded cells in bri1-5R1 as opposed to Ws-2 and bri1-5 (Figure 2H). These results suggest that, unlike stems, the increased pedicel length observed in bri1-5R1 is mostly due to an increase in cell division, possibly aided by a small increase in cell expansion.

bri1-5R1 plants are insensitive to BR:

Because bri1-5R1 plants show reversion of many bri1-5 phenotypes, we tested if bri1-5R1 plants have increased sensitivity to epiBL, as compared to bri1-5 plants. In a root-growth inhibition assay, Ws-2 plants showed a typical response to epiBL, with low concentrations from 0.05 nm to 0.01 μm showing increased primary root length and with concentrations >0.01 μm showing an inhibition of primary root length. Like bri1-5, bri1-5R1 plants showed reduced primary root growth in the absence of exogenous epiBL (Figure 3A). Unlike Ws-2, both bri1-5 and bri1-5R1 showed no inhibition of root growth within the concentrations tested, and at concentrations >0.01 μM, bri1-5R1 plants showed a trend toward increased root growth in response to epiBL treatment (Figure 3A).

Figure 3.—

bri1-5 and bri1-5R1 responses to BRs. Accumulation of BRs is reduced in bri1-5R1 mutants compared to bri1-5 mutants. (A) BR root-growth inhibition for Ws-2, bri1-5, and bri1-5R1 on plates containing 0, 5 × 10⁻⁵, 1 × 10⁻⁴, 5 × 10⁻⁴, 1 × 10⁻³, 5 × 10⁻³, 1 × 10⁻², 5 × 10⁻², 0.1, 0.5, and 1 μm epi-brassinolide measured after 7 days of growth. n > 20 for each data point ± 1 SD. (B) Purification and quantification of sterols and BRs from aerial parts of 5-week-old soil-grown plants carried out according to the method described by Fujioka et al. (2002). See Table S1; for raw data. All values are in nanograms/gram fresh weight.

bri1-5R1 mutants have partially restored levels of brassinosteroid intermediates:

Mutants in many steroid signaling genes have been found to accumulate steroids, suggesting that perception is linked via feedback regulation to biosynthesis (Noguchi et al. 1999; Choe et al. 2002; Wang et al. 2002). To test whether the reversion of the dwarf phenotype of bri1-5 was accompanied by a corresponding change in the regulation of steroid synthesis, we examined the levels of BR biosynthetic intermediates in bri1-5R1 plants, bri1-5 plants, and Ws-2 plants (Figure 3B; supporting information, Table S1). The values for bri1-5 and Ws-2 vary slightly from the values reported previously (Noguchi et al. 1999), although the general pattern is very similar. The differences may be due to alterations in growth conditions. Brassinolide, the end product of the pathway, was still detectable in bri1-5R1 plants, but was reduced approximately sixfold from the level found in bri1-5 plants. bri1-5R1 plants displayed lower levels of 6-oxoBRs than did bri1-5 plants, but around the same levels of 6-deoxoBRs as bri1-5 plants displayed. Since BR signaling represses BR synthesis, we conclude from these results that the bri1-5R1 mutation increases the signaling capacity of BRI1-5.

Our phenotypic results together with our BR measurements suggested that the phenotypes of bri1-5R1 mutants result from increased signaling through BRI1-5. We hypothesized that this increased signaling could be due to (1) ligand-independent activation of BRI1, (2) a bypass of BAK1 function, or (3) re-accumulation of BRI1-5 through ER exit.

BR biosynthesis is required for bri1-5R1 suppression of bri1-5 morphology:

We tested whether or not the phenotypic suppression of dwarfism observed in bri1-5R1 plants was dependent on the availability of BRs. First, we disrupted the BR biosynthetic pathway by using a genetic approach. We constructed strains that were homozygous for bri1-5R1 and for cpd-5 or dwf4-1, loss-of-function alleles for two different enzymes in the BR biosynthetic pathway (Choe et al. 1998; Choe 2004). The triple mutants were indistinguishable from the cpd-5 and dwf4-1 single mutants (Figure 1A), indicating that bri1-5R1 phenotypes do require wild-type BR biosynthesis. Second, we depleted the endogenous pools of BRs by using a chemical inhibitor. We used brassinazole (BRZ), a triazole compound that specifically blocks BR biosynthesis by acting on the product of the DWF4 gene (Asami et al. 2001). We tested whether bri1-5R1 plants were BRZ insensitive by assessing their ability to de-etiolate. bri1-5R1 plants grown in the dark in the presence of 1 μM of BRZ had short hypocotyls and looked like bri1-5-treated seedlings. At the same dose, Ws-2 plants also displayed shorter hypocotyls compared to untreated plants but appeared to be more resistant to BRZ when compared to bri1-5 and bri1-5R1 (Figure 4).

Figure 4.—

BR biosynthesis is required for bri1-5R1 etiolation phenotypes. (A) Morphology of 5-day-old dark-grown seedlings of Ws-2 (wild type) and isogenic bri1-5 and bri1-5R1 grown on half-strength MS medium in the absence (−) or presence (+) of 1 μm BRZ. This experiment is representative of three independent replicates. (B) Lengths of 5-day-old dark-grown seedling hypocotyls in the absence or presence of 1 μm BRZ. Means and standard deviations were calculated from 40 seedlings. This experiment is representative of three independent replicates.

An intact signaling pathway is required for bri1-5R1 suppression of bri1-5 morphology:

To test the relationship between bri1-5R1 phenotypes and BAK1, we crossed bak1-2 into bri1-5R1. Triple mutants for bri1-5R1 and bak1-2 resembled bri1-5 single mutants in stature (Figure 1A, Table 1), indicating that BAK1 is required for the size reversion of bri1-5 to bri1-5R1 and that bri1-5R1 does not act to bypass BAK1. The suppression of the longer bri1-5R1 internodes by bak1-2 is consistent with a requirement for BAK1 in bri1-5R1 reversion of bri1-5. However, the bri1-5R1 long pedicel phenotype was only partially suppressed in the triple bri1-5R1 bak1-2 mutant (Table 1).

These results suggested that bri1-5R1 suppresses bri1-5 phenotypes by increasing signaling through the BR response pathway. To further test the mechanism of suppression by bri1-5R1, we tested whether bin2 mutants could block the morphological suppression mediated by bri1-5R1. The triple mutant bri1-5R1 bin2 plants resembled bin2 plants (Figure 1) or bin2 bri1-5 (Li and Nam 2002) plants, indicating that the mutated BRI1-5R1 receptor signals through BIN2. Thus, the ability of bri1-5R1 to suppress the phenotypes associated with bri1-5 relies on both the availability of BRs and the proper transduction of BR signals.

bri1-5R1 acts as a suppressor by modulating BRI1-5 stability or accumulation:

bri1-5 encodes a partially functional receptor that does not accumulate to wild-type levels because it is retained in the ER where it is subjected to the endoplasmic reticulum–associated degradation (ERAD) process (Hong et al. 2009). Thus, to further investigate the recovery of BRI1-mediated signaling in bri1-5R1 plants, we tested whether the inherent instability of the BRI1-5 protein is suppressed by bri1-5R1. We monitored the accumulation of BRI1 in the Ws-2 accession and in bri1-5 and bri1-5R1 plants by using an affinity-purified anti-BRI1 antibody. BRI1 accumulated to readily detectable levels in Ws-2, but was severely reduced in bri1-5 (Figure 5A). BRI1 accumulation was restored to almost wild-type levels in bri1-5R1 plants, which indicates that the levels of BRI1 are rate limiting for proper BR responses and that bri1-R1 suppresses bri1-5 by allowing the steady-state level of BRI1-5 to increase.

Figure 5.—

BRI1 re-accumulates at the plasma membrane in bri1-5R1. (A) Total protein extracts were prepared from wild-type Ws-2 (wild type) and isogenic bri1-5 and bri1-5R1 plants. These extracts were subjected to an anti-BRI1 protein gel blot. Equal loading for all protein samples was ensured by protein quantification before loading. The nonspecific band detected below BRI1 was used to demonstrate equal loading in each lane after transfer. This experiment is indicative of four independent replicates. (B) Microsomal protein extracts prepared from Ws-2 (wild type) and isogenic bri1-5 and bri1-5R1 plants were treated with either water alone or endoglycosidase H (EndoH; NEB). Samples were collected for anti-BRI1 immunoblot analysis 3 hr after treatment (similar results were seen at 24 hr). Equal loading for all protein samples was ensured by protein quantification of microsomal pellets after resuspension. PM, plasma membrane; ER, endoplasmic reticulum. This experiment is indicative of three independent replicates. (C) Protein samples of bri1-5 plants from B were used separately for protein blot analysis. The samples are overrepresented by threefold and were subjected to higher exposure time. (D) (Top) Morphology of 5-day-old dark-grown seedlings of Ws-2 (wild type) and isogenic bri1-5 and bri1-5R1 grown on half-strength MS medium in the absence (−) or presence (+) of 10 mm kif (Calbiochem, EMD Chem). (Bottom) Lengths of 5-day-old dark- grown seedling hypocotyls grown in the absence or presence of 10 mm kif. The mean and standard deviation were calculated from 30 seedlings. This experiment is representative of two independent replicates.

bri1-5R1 acts as a suppressor by modulating the localization of BRI1-5:

Intuitively, BRI1 exit from the ER, atop general accumulation, is necessary for BR signaling from the cell surface. To assess whether receptor localization contributes to the restoration of BR signaling in bri1-5R1 plants, we monitored BRI1-5R1 exit from the ER by exploiting the fact that BRI1 is a glycoprotein. We used receptor sensitivity to endoglycosidase H (EndoH) as a reporter for putative localization. Typically, plasma membrane-associated glycoproteins are resistant to EndoH digestion whereas ER-retained glycoproteins are sensitive (Hong et al. 2009). We isolated and treated microsomal extracts from wild-type and bri1-5 and bri1-5R1 plants with EndoH. The treated bri1-5 protein extracts produced a single band of high electrophoretic mobility when probed with anti-BRI1 antibodies, consistent with the previously reported ER retention of BRI1-5 (Hong et al. 2009) (Figure 5B). The EndoH treatment of wild-type extracts also produced one single band, but of low electrophoretic mobility. This electrophoretic pattern is consistent with the proper delivery of wild-type receptors to the cell surface. The treatment of bri1-5R1 protein extracts with EndoH produced two bands, indicating that in bri1-5R1 plants the receptor exits the ER, but is also retained in the ER (Figure 5C). While the vast majority of the protein appeared to be EndoH resistant, a significant fraction was EndoH sensitive. This is consistent with a dual localization of the receptor at the ER and at the plasma membrane in bri1-5R1 mutant plants.

Kifunensine does not enhance the ability of bri1-5R1 mutants to elongate:

During the maturation of glycoproteins, the trimming of N-linked glycans acts as a molecular timer that monitors proper protein folding. In the ER and Golgi, terminally misfolded glycoproteins become fully trimmed by 1,2-mannosidases, which targets them for destruction. When these mannosidases are inhibited, for example, by the mannosidase inhibitor Kif, more protein can escape ER quality control and reach the plasma membrane. We believed that this effect would promote a more wild-type receptor distribution in bri1-5R1 plants, allowing us to see whether bri1-5R1 is a hypermorphic mutation. Validating our approach, the Kif treatment of bri1-5 seedlings both lets more BRI1-5 receptor escape the ER and promotes seedling elongation (Figure 5, C and D). However, applying Kif to bri1-5R1 seedlings did not cause additional or faster elongation. Thus, bri1-5R1 does not appear to act as a gain-of-function in the control of the de-etiolation process mediated by the BR signaling pathway.

DISCUSSION

The binding of BR to the LRRs of BRI1 most likely occurs on the cell surface. This cell surface receptor–ligand interaction activates a fairly well-defined intracellular signal transduction cascade that primarily promotes cell elongation. The analysis of bri1 mutants blocked at successive steps of the secretory pathway, such as bri1-5 and bri1-9, has shown that BRI1 abundance at the cell surface is critical for optimal BR perception and hence for BR signaling. The delivery of newly synthesized BRI1 receptors to the plasma membrane is controlled by the activity of at least three independent, but interrelated, protein-folding quality control pathways. Here, we report that the aberrant accumulation and cellular distribution of the BRI1-5 protein, which is in abnormally low quantities at the plasma membrane and largely accumulates in the ER, is restored to almost wild-type levels by an intragenic suppressor mutation that we named bri1-R1. We show that the restoration of both protein levels and protein localization is correlated with the suppression of the morphological phenotypes associated with bri1-5. Interestingly, this intragenic reversion also allows discrete but important organ-specific gain-of-function phenotypes to take place. We propose that bri1-R1 suppresses bri1-5 (1) by antagonizing the activities of three ER resident protein-folding pathways by an unknown mechanism and (2) by increasing the signaling activity of the BR pathway above the wild-type threshold in specific cell types.

LRRs as a mutational target in BRI1:

In bri1-5, the second Cys residue of the N-terminal conserved Cys pair is substituted by a Tyr (C69Y). This mutation is associated with morphological phenotypes reminiscent of BR biosynthetic mutants, indicating that these cysteines play an important role in vivo. The Cys capping domains are a feature common to many plant extracellular LRR proteins. Initially, this pair of cysteines was proposed to be involved in the formation of disulfide bonds that aid in homo- or heterodimerization of receptor-like kinases. This model remains attractive yet speculative (Dievart and Clark 2003). An emerging model, based mainly on in vitro folding studies, suggests that these Cys pairs are involved in forming specific disulfide bonds to help nucleate and stabilize the fold of the first LRR into an energetically favorable conformation. Consistent with this model, the C69Y mutation in bri1-5 forces BRI1 to localize in the ER because of its improper folding. Here, we demonstrate that the mutation of a conserved glycine to a glutamic acid (bri1-R1; G87E) located in the closest LRR to the amino-terminal paired cysteines largely compensates for bri1-5 defects. On the basis of a comparative and quantitative morphometric analysis that we conducted on wild-type, bri1-5, and bri1-5R1 plants (Table 1), we show that most phenotypes of bri1-5R1 plants are intermediate between bri1-5 and wild-type plants (Table 1), consistent with the second-site mutation restoring receptor localization and not enhancing receptor activity.

Little is known about the functions of the LRRs in BRI1. In contrast, there are >15 mutations isolated in the smaller kinase domain and ∼10 in the island domain, which is 70 amino acids long (Li and Chory 1997; Noguchi et al. 1999; Friedrichsen et al. 2000). Thus, in formal genetic terms, the LRRs appear to be almost dispensable for function. The only other LRR missense mutation identified in BRI1 is bri1-9, a weak mutant caused by a missense mutation in the 22nd LRR (Figure 1, B and C; Noguchi et al. 1999). Thus, bri1-R1 is the second mutation found by forward genetics in the LRRs of BRI1, which comprise more than half of the total length of the BRI1 protein. It is likely that additional missense mutations in LRRs have not been obtained because they confer weak loss-of function phenotypes that would have been missed in the stringent genetic screening conducted to date. Furthermore, it is reasonable to speculate that gain-of-function properties could be revealed only in sensitized genetic backgrounds such as the bri1-5 and bri1-9 mutants. Clear evidence for direct LRR–ligand interactions is known for at least two other LRR–RLK plant perception systems, CLAVATA1 (CLV1) and FLAGELLIN SENSITIVE-2 (FLS2), which, respectively, play roles in the maintenance of stem cells and plant immunity. However, the role of the LRR module in these perception systems is rather elusive. Interestingly, mutations in LRRs do not confer similar properties; the identical amino acid substitution to elg, an activated variant of BAK1, in an LRR of CLV1 causes a dominant negative phenotype (Dievart et al. 2003; Whippo and Hangarter, 2005) (Figure 1B). Thus, similar mutations in similar LRRs can translate into radically opposed effects. The identification of bri1-R1 as an intragenic revertant localized in the canonical LRR module of BRI1 is quite unique. We present here the first report of an intragenic reversion in a member of the LRR–RLK superfamily. As such, our findings have implication for a better understanding of LRR–RLK signaling pathways in plants.

Identical or similar mutations to bri1-R1 occur as natural polymorphic variants in several plant species:

In Arabidopsis, the glycine residue mutated in bri5-R1 is remarkably invariant in other members of the BR receptor family (BRL1 and BRL3) as well as in other LRR–RLKs, including BAK1, SERK1, FLS2, and CLV1 (Figure 1D). Orthologs of BRI1 have been isolated from many plant species from dicots such as tomato to monocots such as rice, barley, and wheat. Strikingly, with the exception of Arabidopsis, it seems that other plant species have evolved BR receptors in which the conserved Gly residue affected by bri1-R1 is substituted for an Asp or a Glu residue. Thus, our intragenic suppressor, which changes the same amino acid as found in the OsBRI1 sequence, offers a snapshot of genetic variation in phylogenetically related BR receptors. The potentially hypermorphic effects observed in bri1-5R1 plants (Table 1) are not inconsistent with an adaptive phenomenon where the genetic assimilation of an Asp or a Glu, instead of a Gly, at a specific position would allow environmental information to be embodied in new traits. It is tempting to speculate that this genetic variation would account, to some extent, for changes in the ability of various plants to elongate. Whether the latter is relevant for the functional diversification of BR receptors in land plants is an open question that needs to be experimentally tested.

The morphology of bri1-5R1 plants is suggestive of gain-of-function phenotypes:

The amino acid sequence analysis of BRI1 orthologs revealed that the bri1-R1 mutation occurs as a natural variant in many plant species. Here we demonstrate that the bri1-R1 mutation is able to restore BR-signaling responses to plants with the weak bri1-5 mutation. Our analysis of bri1-5 and bri1-5R1 morphologies revealed three classes of phenotypes. The first class appeared to be bri1-5 specific (the additional valves in the fruit), while the second class was already present in bri1-5 and enhanced in bri1-5R1 (the increased pedicel length and the extra anthers). The third class is bri1-5R1 specific, and as such, represents putative gain-of-function phenotypes (more sepals and petals). We anticipate that if bri1-R1 indeed is a gain-of-function mutant, it would display phenotypes similar to those found in plants overexpressing BRI1 or other positive regulators of BR signaling, including longer leaves and dramatically elongated petioles. On the other hand, it is possible that the phenotypes specific to bri1-R1 in the bri1-5 genetic background will simply be more pronounced in a wild-type background. Interestingly, activation-tagging screens have identified several dominant extragenic suppressors of bri1-5, including BAK1, BRL1, and BRS1 (Li et al. 2001, 2002; Zhou et al. 2004). The morphometric analysis of bri1-5R1 plants revealed that, on average, traits that are affected in bri1-5R1 such as pedicel length, leaf width, and sepal and petal number were not altered in the extragenic suppressors. Thus, in comparison to these extragenic suppressors, the intragenic suppressor, bri1-R1, appears to act differently, perhaps by modulating the signaling competency of BRI1. The phenotypic analysis of transgenic plants in which a variant of BRI1 harboring the bri1-R1 mutation but without bri1-5 transformed into bri1-null plants will help resolve this issue.

bri1-5R1 further implicates the BR-signaling pathway in the control of the plant cell cycle:

The increased size of bri1-5R1 plants compared to bri1-5 plants appeared to be due to an increase in the lengths of internodes. To gain insight into the physiological and cellular mechanisms underlying the reversion of the phenotypes associated with bri1-5, we performed a histological analysis. Our transverse stem sections indicate that the suppression of the morphological phenotypes is driven by a significant increase in cell elongation (Figure 2A). The latter was expected because BRs act on plant development mainly by controlling the ability of a cell to elongate. To our surprise, we noted that the increase in pedicel length in bri1-5R1 plants was due to an increase in cell division possibly aided by a small contribution from cell elongation (Figure 2, B and C). Our results demonstrate that the BR-signaling pathway is able to modulate the cell cycle, although we do not know where the input from BR signaling connects to cell cycle regulation.

bri1-5R1 restores the flux of the BR biosynthetic and signaling pathway concomitantly:

The perception of BRs by BRI1 at the cell surface alters the expression of hundreds of genes through the balanced activities of BES1 and BZR1, two plant-specific transcription factors. A significant subset of these BR responsive genes encodes key enzymes required for the proper biosynthesis of BRs. There at least two epistatic read-outs available to measure the signaling flux of the BR-signaling pathway: (1) the dephosphorylation of BES1 and BZR1 and (2) the feedback regulation of the biosynthetic pathway by the signaling pathway. To coordinate BR homeostasis and signaling, BZR1 represses the expression of the BR biosynthetic genes CPD and DWF4 by directly binding to their promoters. Accordingly, when signaling occurs, the transcription of CPD and DWF4 is dramatically reduced by the action of BZR1. Thus, the activation of signaling can be monitored by measuring the transcriptional activities of DWF4 and/or CPD. Here, we measured the activation of the BR-signaling pathway by monitoring the flux of the BR biosynthetic pathway, the most downstream and relevant read-out of the feedback regulatory loop. As previously shown, our BR quantification showed the increased accumulation of BR intermediates in bri1-5 plants (Figure 3B), an indication of increased BR metabolism and hence of reduced signal transduction. On average, the BR metabolic pathway in bri1-5R1 plants was almost identical to that of wild-type plants. We assume that the restoration of wild-type endogenous BR accumulation in bri1-5R1 is probably achieved through the successful perception and transduction of BR signals. Our BR measurements did not allow us to assess whether bri1-5R1 was a real gain-of-function because we measured the bulk accumulation of BRs in whole aerial parts of plants, and our measurements lacked the sensitivity required to detect tissue-specific variation in BR pools. This is consistent with the discrete but organ-specific gain-of-function phenotypes associated with the bri1-5R1 mutation. For the same reason, we anticipate that the phosphorylation status of BES1 and/or the expression of CPD or DWF4 would have been as informative as our BR measurements in monitoring the localized BR responses in actively growing tissues of bri1-5R1 plants. The analysis of organ-specific expression patterns of the additional components of the BRI1 signaling pathway will be important for uncovering the mechanisms responsible for these phenotypes.

bri1-5R1 antagonizes the activities of the unfolded protein response:

The Unfolded Protein Response (UPR) is a system capable of discriminating native and nonnative proteins by recognizing several common features of improperly/incompletely folded proteins, such as unpaired Cys residues, immature Asn-linked glycans, and exposed hydrophobic amino acids. The UPR is mediated by both the endoplasmic reticulum–mediated quality control (ERQC) and the ERAD machineries. Our endoglycosidase-H digests, together with our ERAD inhibition assays using Kif, are consistent with previous reports showing that BRI1-5 is immaturely glycosylated and retained intracellularly by a functional checkpoint. Our findings show that bri1-R1 restores the functionality of the BR-signaling pathway probably by promoting the escape of the BRI1 receptor from the ER. This scenario has three consequences: (1) the mutant BRI1 is no longer subject to the ERAD machinery and re-accumulates to almost wild-type levels, (2) terminal glycosylation can proceed further in the Golgi apparatus and, consequently, (3) the receptor re-accumulates at the cell surface. It is unclear how this intragenic mutation allows the delivery of a defective receptor from the ER to the cell surface. Our first hypotheses put forth a model in which bri1-R1 compensates for the structural defects associated with the bri1-5 mutation. This compensation subsequently allows the successful bypass of the Bip-dependent, the Thiol-mediated, and the UGGT-based retention mechanisms, which are fundamental to the ERQC activities. Because the perturbation of only one of these processes is necessary and sufficient for ER retention, our findings indicate that a single amino acid substitution in the first LRR of BRI1 is capable of antagonizing the activity of three protein quality control pathways.

The genetic interaction between bri1-5R1 and BAK1 is consistent with our former hypothesis. We cannot exclude models in which bri1-R1 acts as a suppressor by increasing interactions within the BRI1 extracellular domain, between BRI1 monomers, or between BRI1 and BAK1. Because BRI1 is able to associate with itself and BAK1, we speculate that the so-called secondary quality control of the ER could be involved in the ER retention of BRI1-5. The secondary quality control ensures the proper oligomerization of multisubunit receptors in the ER via a mechanism involving the coatamer proteins (COPI). While highly speculative, it is possible that the assembly of BRI1 and BAK1 higher-order complexes is monitored at the ER level. The oligomeric BR receptors assembled in the proper conformation could escape the ER, whereas improper conformations created by unpaired cysteine residues will result in weaker oligomeric interactions and subsequent ER retention. In bri1-5R1 plants, a stronger interaction between the ECDs of BAK1 and BRI1 could more efficiently bury the structural defects of BRI1-5, allowing more efficient relocalization to the plasma membrane. In the process, a more efficient BRI1/BAK1 oligomer would be expressed at the cell surface where it could enhance the signaling competency of the pathway. Analysis of the physical interaction between BRI1 monomers and BRI1 and BAK1, perhaps with the bri1-5R1 mutation, will be required to ultimately determine the extracellular interactions of these two receptor kinases.