skip to main content


Title: Genetic Interactions Between BEN1 ‐ and Cytochrome P450‐Mediated Brassinosteroid Inactivation
Abstract

Brassinosteroids (BRs) are essential plant growth‐promoting hormones involved in many processes throughout plant development, from seed germination to flowering time. SinceBRsdo not undergo long‐distance transport, cell‐ and tissue‐specific regulation of hormone levels involves both biosynthesis and inactivation. To date, tenBR‐inactivating enzymes, with at least five distinct biochemical activities, have been experimentally identified in the model plantArabidopsis thaliana. Epigenetic interactions betweenT‐DNAinsertion alleles and genetic linkage have hindered analysis of higher‐order null mutants in these genes. A previous study demonstrated that thebas1‐2 sob7‐1 ben1‐1triple‐null mutant could not be characterized due to epigenetic interactions between the exonicT‐DNAinsertions inbas1‐2andsob7‐1,causing the intronicT‐DNAinsertion ofben1‐1to revert to a partial loss‐of‐function allele. We usedCRISPR‐Cas9genome editing to avoid this problem and generated thebas1‐2 sob7‐1 ben1‐3triple‐null mutant. This triple‐null mutant resulted in an additive seedling long‐hypocotyl phenotype. We also uncovered a role forBEN1‐mediatedBR‐inactivation in seedling cotyledon petiole elongation that was not observed in the singleben1‐2null mutant but only in the absence of bothBAS1andSOB7. In addition, genetic analysis demonstrated thatBEN1does not contribute to the early‐flowering phenotype, whichBAS1andSOB7redundantly regulate. Our results show thatBAS1,BEN1,andSOB7have overlapping and independent roles based on their differential spatiotemporal tissue expression patterns

 
more » « less
NSF-PAR ID:
10483870
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Physiologia Plantarum
Volume:
176
Issue:
1
ISSN:
0031-9317
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between threeMechanosensitive channel ofSmall conductance‐Like (MSL) proteins fromArabidopsis thaliana.MSLproteins areArabidopsishomologs of the bacterialMechanosensitivechannel ofSmall conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock.MSL1 localizes to the inner mitochondrial membrane, whileMSL2 andMSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion inMSL1, both in wild type and inmsl2 msl3mutant backgrounds.msl1single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling inmsl2 msl3double mutants was exacerbated in themsl1 msl2 msl3triple mutant. However, the introduction of themsl1lesion into themsl2 msl3mutant background suppressed othermsl2 msl3mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version ofMSL1. In yeast‐based interaction studies,MSL1 interacted with itself, but not withMSL2 orMSL3. These results establish that the abnormalities observed inmsl2 msl3double mutants is partially dependent on the presence of functionalMSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

     
    more » « less
  2. Abstract

    Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many different levels. Recent research has revealed that stress‐responsive NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factorRD26 is regulated byBRsignaling and antagonizesBES1 in the interaction between growth and drought stress signaling. However, the upstream signaling transduction components that activateRD26 during drought are still unknown. Here, we demonstrate that the function ofRD26 is modulated byGSK3‐like kinaseBIN2 and protein phosphatase 2CABI1. We show thatABI1, a negative regulator inabscisic acid (ABA)signaling, dephosphorylates and destabilizesBIN2 to inhibitBIN2 kinase activity.RD26 protein is stabilized byABAand dehydration in aBIN2‐dependent manner.BIN2 directly interacts and phosphorylatesRD26in vitroandin vivo.BIN2 phosphorylation ofRD26 is required forRD26 transcriptional activation on drought‐responsive genes.RD26 overexpression suppressed the brassinazole (BRZ)  insensitivity ofBIN2 triple mutantbin2 bil1 bil2, andBIN2 function is required for the drought tolerance ofRD26 overexpression plants. Taken together, our data suggest a drought signaling mechanism in which drought stress relievesABI1 inhibition ofBIN2, allowingBIN2 activation. Sequentially,BIN2 phosphorylates and stabilizesRD26 to promote drought stress response.

     
    more » « less
  3. Summary

    A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth‐promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode‐specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild‐type petioles was identified through a forward genetic screen. In addition to stem‐specific elongation, this mutant, namedtomato internode elongated ‐1(tie‐1) is more sensitive to theGAbiosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactiveGAs compared with wild‐type plants. The mutation responsible for the internode elongation phenotype was mapped toGA2oxidase 7, a classIII GA2‐oxidase in theGAbiosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinantTIEprotein was shown to have bona fideGA2‐oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role ofGAbiosynthetic genes in organ‐specific elongation.

     
    more » « less
  4. Abstract Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA–protein and protein–protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation. 
    more » « less
  5. Summary

    Plant smallRNAs (sRNAs) modulate key physiological mechanisms through post‐transcriptional and transcriptional silencing of gene expression. SmallRNAs fall into two major categories: those are reliant onRNA‐dependentRNApolymerases (RDRs) for biogenesis and those that are not. KnownRDR1/2/6‐dependentsRNAs include phased and repeat‐associated short interferingRNAs, while knownRDR1/2/6‐independentsRNAs are primarily microRNAs (miRNA) and other hairpin‐derivedsRNAs. In this study we produced and analyzedsRNA‐seq libraries fromrdr1/rdr2/rdr6triple mutant plants. We found 58 previously annotated miRNAloci that were reliant onRDR1, ‐2, or ‐6function, casting doubt on their classification. We also found 38RDR1/2/6‐independentsRNAloci that are notMIRNAs or otherwise hairpin‐derived, and did not fit into other known paradigms forsRNAbiogenesis. These 38sRNA‐producing loci have as‐yet‐undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein‐coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types ofsRNAinArabidopsis thaliana.

     
    more » « less