Loss-of-Function Mutations in the Arabidopsis Heterotrimeric G-protein Subunit Enhance the Developmental Defects of Brassinosteroid Signaling and Biosynthesis Mutants (original) (raw)
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Scientific Reports
Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization. Steroid hormones are bioactive substances that are widely conserved in eukaryotes 1 and serve as signaling molecules to control growth and development. The plant steroid hormones known as brassinosteroids (BRs) play important roles in plant development and in responses to environmental cues, such as cell elongation, cell division, xylem development, stresses and pathogen resistance 2-6. BRs are recognized by the BR receptor brassinosteroid insensitive 1 (BRI1), a Ser/Thr kinase, which has a leucine-rich repeat (LRR) domain and resides on the plasma membrane and endosomes. The dwarf phenotype of BRI1-deficient mutants (bri1) 7 suggests that BRI1 plays an important role in plant growth. The plasma membrane proteins BRI1-associated receptor kinase 1 (BAK1) 8, 9 , BR-signaling kinase 1 (BSK1) 10 , and BRI1 kinase inhibitor 1 (BKI1) 11 are involved in BR signaling by associating with BRI1. BRI1 is localized not only to plasma membranes but also to endosomes 12. The requirement that ligand-bound receptors, including innate immunity-related Toll-like receptors (TLRs), be localized to endosomes has been widely observed in animals 13. BRI1 endocytosis is regulated by the guanine nucleotide exchange factor for ADP-ribosylation factor (ARF-GEF) GNOM 14 , adaptor protein complex-2 (AP-2) 15 , and plant-specific TPLATE adaptor complex (TPC), which is associated with key components of clathrin-mediated endocytosis (CME) 16. BRI1 is considered to translocate to the vacuoles through processes that are regulated by ubiquitination 17 and
Cell, 2002
BL), the most active BR (Mussig and Altmann, 1999), genetic studies have been less illuminating in identifying components of the BR signaling pathway. largely in one gene, BRI1, which encodes a critical com-Biology Laboratory ponent of the BR receptor (Clouse et al., 1996; Kausch-The Salk Institute for Biological Studies mann et al., 1996; Li and Chory, 1997). Unlike animal La Jolla, California 92037 steroid nuclear receptors that directly activate target 2 Department of Molecular, Cellular, and gene expression upon ligand binding (Beato and Klug, Developmental Biology 2000; Mangelsdorf et al., 1995), BR signals are perceived University of Michigan at the cell surface by the plasma membrane localized Ann Arbor, Michigan 48109 BRI1, a member of the leucine-rich repeat (LRR) receptor 3 Plant Functions Lab kinase family (Friedrichsen et al., 2000; Li and Chory, RIKEN (The Institute of Physical and Chemical 1997; Wang et al., 2001). BRI1 is composed of an extra-Research) cellular domain containing 25 LRRs interrupted by a Wako-shi, Saitama 351-0198 70 amino acid island domain, a single transmembrane Japan domain, and an intracellular serine/threonine kinase domain (Li and Chory, 1997). A chimeric receptor composed of the BRI1 extracellular domain and the kinase Summary domain of Xa21, a rice LRR receptor kinase for disease resistance, confers BL-dependent pathogen responses Plant steroid hormones, known as brassinosteroids to rice cells (He et al., 2000). In addition, both membrane (BRs), signal through a plasma membrane localized fractions and immunoprecipitates containing BRI1 bind receptor kinase BRI1. We identified bes1, a semidomi-3 H-labeled BL specifically, and such binding is greatly nant suppressor of bri1, which exhibits constitutive reduced in plants harboring mutations in the extracellu-BR response phenotypes including long and bending lar domain (Wang et al., 2001). BRI1's kinase domain petioles, curly leaves, accelerated senescence, and displays serine/threonine kinase activity in vitro, and BL constitutive expression of BR-response genes. BES1 treatment in plants induces BRI1 autophosphorylation accumulates in the nucleus in response to BRs. BES1 (Friedrichsen et al., 2000; Oh et al., 2000; Wang et al., is phosphorylated and appears to be destabilized by 2001). Thus, all available evidence strongly implicates the glycogen synthase kinase-3 (GSK-3) BIN2, a nega-BRI1 as a critical component of the BR-receptor comtive regulator of the BR pathway. These results estabplex, which likely transduces the BR signal to downlish a signaling cascade for BRs with similarities to the stream targets through its kinase domain.
Loss of Function in GIGANTEA Gene is Involved in Brassinosteroid Signaling
2011
Brassinosteroids (BRs) are plant steroid hormones that play essential roles in growth and development. Mutations in BR-signaling pathways cause defective in growth and development like dwarfism, male sterility, abnormal vascular development and photomorphogenesis. Transition from vegetative to reproductive growth is a critical phase change in the development of a flowering plant. In a screen of activation-tagged Arabidopsis, we identified a mutant named abz126 that displayed longer hypocotyls when grown in the dark on MS media containing brassinazole (Brz), an inhibitor of BRs biosynthesis. We have cloned the mutant locus using adapter ligation PCR walking and identified that a single T-DNA had been integrated into the ninth exon of the GIGANTEA (GI) gene, involved in controling flowering time. This insertion resulted in loss-of-function of the GI gene and caused the following phenotypes: long petioles, tall plant height, many rosette leaves and late flowering. RT-PCR assays on abz1...
Differential expression of the brassinosteroid receptor-encoding BRI1 gene in Arabidopsis
Planta, 2014
the wild phenotype, expression from the photosynthesisassociated CAB3 and the vasculature-specific SUC2 and ATHB8 promoters resulted in plants with varying morphogenic defects. Our results reveal complex differential regulation of BRI1 expression, and suggest that by influencing the distribution and abundance of the receptor this regulation can enhance or attenuate BR signalling.
The Plant cell, 2015
Brassinosteroids (BRs) play important roles in plant development and the response to environmental cues. BIL1/BZR1 is a master transcription factor in BR signaling, but the mechanisms that lead to the finely tuned targeting of BIL1/BZR1 by BRs are unknown. Here, we identified BRZ-SENSITIVE-SHORT HYPOCOTYL1 (BSS1) as a negative regulator of BR signaling in a chemical-biological analysis involving brassinazole (Brz), a specific BR biosynthesis inhibitor. The bss1-1D mutant, which overexpresses BSS1, exhibited a Brz-hypersensitive phenotype in hypocotyl elongation. BSS1 encodes a BTB-POZ domain protein with ankyrin repeats, known as BLADE ON PETIOLE1 (BOP1), which is an important regulator of leaf morphogenesis. The bss1-1D mutant exhibited an increased accumulation of phosphorylated BIL1/BZR1 and a negative regulation of BR-responsive genes. The number of fluorescent BSS1/BOP1-GFP puncta increased in response to Brz treatment, and the puncta were diffused by BR treatment in the root a...
Oligomerization between BSU1 Family Members Potentiates Brassinosteroid Signaling in Arabidopsis
Molecular Plant, 2015
Brassinosteroid (BR) regulates diverse physiological and developmental processes in plants (Choudhary et al., 2012). BR binds to the receptor kinase BRI1 to trigger a phosphorylation/ dephosphorylation cascade, which includes phosphorylation of Brassinosteroid Signaling Kinase 1 (BSK1) and Constitutive Differential Growth 1 (CDG1) by BRI1, phosphorylation of BSU1 by CDG1, and dephosphorylation of BIN2 (a GSK3-like kinase) by BSU1 (Kim et al., 2009, 2011; Wang et al., 2014). Almost all the downstream components of BR signaling are encoded by multiple genes of a family. The Arabidopsis BSU1 family (BSUf; BSU1, BSL1, BSL2, and BSL3) is classified into a unique phosphatase family, the PPKL (Protein Phosphatases with Kelch-Like repeat domains), which is not found in animals (Kutuzov and Andreeva, 2002; Mora-Garcia et al., 2004). BSUf members contain an N-terminal Kelch-repeat domain and a C-terminal phosphatase domain that is closely related to protein phosphatase 1. Knockdown of BSL2 and BSL3 by artificial micro-RNA in the bsu1bsl1 mutant (amiBSL2,3/bsu1bsl1) causes pleiotropic defects, including severe dwarfism similar to strong BR-deficient or BR-insensitive mutants and abnormal stomatal development (Kim et al., 2009, 2012; Youn et al., 2013). A recent study suggested that BSUf members exhibit specific/non-overlapping functions in addition to BR signaling, although all four members are involved in BR signaling (Maselli et al., 2014). However, the biochemical characteristics underlying the functional redundancy and diversity of PPKL family members are largely unknown.
Plant & cell physiology, 2014
Heterotrimeric G-proteins, comprised of a, b and g subunits, are important signal transducers across phyla. The G-proteins are well characterized in the model plants Arabidopsis and rice, and their homologs are identified in a few other plant species; however, information about the roles played by G-proteins in regulating various growth and developmental traits particularly from polyploid crops is still awaited. In this study, we have isolated one Ga (BniB.G1), three Gb (BniB.G1-BniB.G3) and four Gg (BniB.G1-BniB.G4) coding sequences from the paleopolyploid Brassica nigra, a major condiment crop of the Brassicaceae family. Sequence and phylogenetic analysis revealed that whole-genome triplication events in the Brassica lineage had proportionally increased the inventory of the Gb subunit, but not of the Ga and Gg subunits in B. nigra. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that members of the G-protein subunit genes have distinct temporal and spatial expression patterns and were differentially altered in response to various stress and phytohormone treatments, thereby suggesting differential transcriptional regulation of G-protein genes in B. nigra. Interestingly, specific members of G-protein subunits were co-expressed across plant developmental stages, and in response to different elicitor treatments. Yeast-based interaction screens further predicted that the B. nigra G-protein subunits interacted in most of the possible combinations, although showing a high degree of interaction specificity between different G-protein subunits. Our data on physical interactions coupled with the co-expression pattern of the multiple G-protein subunit genes suggested that tissue-and condition-specific functional combinations of Gabg heterotrimers may exist in paleopolyploid B. nigra, to control diverse growth and development processes.
Genes & Development, 2004
Mutant analysis has been tremendously successful in deciphering the genetics of plant development. However, less is known about the molecular basis of morphological variation within species, which is caused by naturally occurring alleles. In this study, we succeeded in isolating a novel regulator of root growth by exploiting natural genetic variation in the model plant Arabidopsis. Quantitative trait locus analysis of a cross between isogenized accessions revealed that a single locus is responsible for ∼80% of the variance of the observed difference in root length. This gene, named BREVIS RADIX (BRX), controls the extent of cell proliferation and elongation in the growth zone of the root tip. We isolated BRX by positional cloning. BRX is a member of a small group of highly conserved genes, the BRX gene family, which is only found in multicellular plants. Analyses of Arabidopsis single and double mutants suggest that BRX is the only gene of this family with a role in root development. The BRX protein is nuclear localized and activates transcription in a heterologous yeast system, indicating that BRX family proteins represent a novel class of transcription factors. Thus, we have identified a novel regulatory factor controlling quantitative aspects of root growth.
Developmental Cell, 2002
response (Clouse et al., 1996; Li and Chory, 1997). BRI1 is a leucine-rich repeat (LRR) receptor kinase, with an Plant Biology Laboratory The Salk Institute for Biological Studies extracellular domain containing 25 LRRs and a 70 amino acid island motif between the 21 st and 22 nd LRRs, a 10010 North Torrey Pines Road La Jolla, California 92037 transmembrane domain, and a cytoplasmic kinase domain (Li and Chory, 1997). BRI1 is localized to the plasma 2 Department of Plant Biology Carnegie Institution of Washington membrane of growing cells, where it functions as a critical component of the BR receptor (Friedrichsen et al., 260 Panama Street Stanford, California 94305 2000; He et al., 2000; Wang et al., 2001). Biochemical studies have shown that brassinolide (BL), the most 3 Plant Functions Laboratory RIKEN (The Institute of Physical and Chemical active BR, binds to BRI1 and activates its kinase activity in Arabidopsis plants (Wang et al., 2001). BL binding Research) Wako-shi, Saitama 351-0198 and subsequent kinase activation is abolished by a mutation in the 70 amino acid island motif of the extracellu-Japan lar domain. Thus, BRI1 perceives the BR signal through its extracellular domain and initiates a signal transduction cascade through its cytoplasmic kinase activity. Summary How the BR signal is transduced from BRI1 to the nucleus remains unclear. Recent molecular genetic studies Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but have implicated a glycogen synthase kinase 3-like protein (BIN2) as a negative regulator of BR responses (Li how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identi-et al., 2001; Li and Nam, 2002). The precise role of BIN2 in the signaling pathway is still not known. fication of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation