BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana

Abstract

Brassinosteroid-insensitive 1 (BRI1) of Arabidopsis thaliana encodes a cell surface receptor for brassinosteroids. Mutations in BRI1 severely affect plant growth and development. Activation tagging of a weak bri1 allele (bri1-5) resulted in the identification of a new locus, brs1-1D. BRS1 is predicted to encode a secreted carboxypeptidase. Whereas a brs1 loss-of-function allele has no obvious mutant phenotype, overexpression of BRS1 can suppress bri1 extracellular domain mutants. Genetic analyses showed that brassinosteroids and a functional BRI1 protein kinase domain are required for suppression. In addition, overexpressed BRS1 missense mutants, predicted to abolish BRS1 protease activity, failed to suppress bri1-5. Finally, the effects of BRS1 are selective: overexpression in either wild-type or two other receptor kinase mutants resulted in no phenotypic alterations. These results strongly suggest that BRS1 processes a protein involved in an early event in the BRI1 signaling.

Figure 1

BRS1 suppresses multiple bri1-5 defects. (A) Comparison of plant phenotypes of wild-type plants (WT, ecotype: Ws-2), bri1-5, and bri1-5 brs1-1D 16 days after germination. Scale bar = 1 cm. (B) Physiological characteristics of WT, bri1-5, and bri1-5 brs1-1D. Measurements were taken 35 days after germination (except flowering time measurements). The means (± SD) were from the measurements of 40 plants per genotype.

Figure 2

The BRS1 gene encodes a protein with homology to serine carboxypeptidases (54% and 53% identity with wheat and barley serine carboxypeptidase II proteins; 28% identity with yeast Kex1p protein). (A) The flanking sequence of the T-DNA was cloned via inverse PCR. The T-DNA insert localizes to the bottom part of chromosome IV. 5′ of the T-DNA, 6.5 kb from the 4 × 35S enhancers, there is a gene encoding a signal recognition particle receptor-like protein. 3′ of the T-DNA, 1.1 kb from 4 × 35S enhancers, there is a gene encoding a serine carboxypeptidase, which was subsequently confirmed as the suppressor, BRS1. (B) Comparison of the cDNA and genomic sequences indicated that BRS1 has 9 exons and 8 introns. (C) Deduced amino acid sequence of BRS1. A possible signal peptide cleavage site is indicated by an arrow. Five potential N-linked glycosylation sites are marked in the open boxes. The asterisks below an amino acid indicate the three putative “catalytic triad” amino acids, S181, D386, and H438. A possible cleavage linker peptide is underlined. The BRS1 sequence was obtained from GenBank (accession no. AL161577, reference GI: 7269962).

Figure 3

A Northern blot shows that the expression of BRS1 in bri1-5 brs1-1D is elevated compared with that in wild-type Ws-2 and bri1-5 plants. Ten micrograms total RNA from 4-week-old above ground tissues was loaded in each lane. The blot was hybridized with a ³²P-labeled BRS1 cDNA. ACT7 cDNA was used as a probe to show equal loading.

Figure 4

Transformation of BRS1 cDNA driven by a CaMV 35S constitutive promoter into bri1-5 recapitulated the mutant suppression phenotype. (A) Wild-type Ws-2 plants. (B) bri1-5 plants. (C) bri1-5 brs1-1D plants. (D) Transgenic plants with a BRS1 cDNA construct confirmed that BRS1 suppresses the bri mutant phenotype. Plants were grown for 35 days under continuous illumination. Scale bar = 2 cm.

Figure 5

BRS1 selectively regulates BRI1 signaling pathway. Wild-type plants harboring the brs1-1D allele (WT brs1-1D) do not show phenotypic alterations. (A) Phenotypes of a wild-type (WT, Ws-2) and a WT brs1-1D plant (both are 18 days old). Size bar = 1 cm. (B) Average main stem length of 37-day WT and WT brs1-1D plants under continuous illumination condition. The means (± SD) were from measurements of 28 plants per genotype. (C) The expression levels of BRS1 in WT and WT brs1-1D plants. ACT7 was used to show equal loading of total RNA.

Figure 6

Biosynthesis of BR is required for BRS1 regulation. BRS1 failed to suppress mutants containing homozygous BR-deficient loci, dwf4-1. The mutants were generated through genetic crossing. The genotypes were determined by both PCR analyses and DNA sequencing. bri1-5 dwf4-1 double mutant plants are phenotypically identical to bri1-5 dwf4-1 brs1-1D triple mutant plants. The plants were photographed 56 days after germination. Scale bar = 1 cm.