Brassinosteroid Signal Transduction Research Articles

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.

A Small G Protein as a Novel Component of the Rice Brassinosteroid Signal Transduction

Brassinosteroids (BRs) are a class of steroid hormones that are essential for plant growth and development. The BR signal transduction pathway in the dicot model plant Arabidopsis is well established, but the components connecting the BR signaling steps in rice have not been fully explored. For example, how the BR signaling is fine-tuned in rice, especially at the BR receptor level, is largely unknown. Here we show that OsPRA2, a rice small G protein, plays a repressive role in the BR signaling pathway. Lamina inclination, coleoptile elongation, and root inhibition assays indicated that rice plants with suppressed expression of OsPRA2 were more sensitive to exogenously applied brassinolide than the wild-type plants. Conversely, rice overexpressing OsPRA2 was less sensitive to exogenous brassinolide. Further study uncovered that OsPRA2 inhibited the dephosphorylation of, and thus inactivated the transcription factor BRASSINAZOLE-RESISTANT 1 (OsBZR1). More importantly, OsPRA2 was found to co-localize with and directly bind to rice BR receptor BRASSINOSTEROID-INSENSITIVE 1 (OsBRI1) at the plasma membrane. Additionally, the in vitro assays showed that OsPRA2 inhibits its autophosphorylation. This OsPRA2–OsBRI1 interaction led to the dissociation of OsBRI1 from its co-receptor OsBAK1, and abolished OsBRI1-mediated phosphorylation of OsBAK1. Together, these results reveal a possible working mechanism of OsPRA2 as a novel negative regulator on OsBRI1 and OsBZR1 and extend the knowledge about the regulatory mechanism of rice BR signaling.

Involvement of Auxin and Brassinosteroid in Dwarfism of Autotetraploid Apple (Malus × domestica).

The plant height is an important trait in fruit tree. However, the molecular mechanism on dwarfism is still poorly understood. We found that colchicine-induced autotetraploid apple plants (Malus × domestica) exhibited a dwarf phenotype. The vertical length of cortical parenchyma cells was shorter in autotetraploids than in diploids, by observing paraffin sections. Hormone levels of indoleacetic acid (IAA) and brassinosteroid (BR) were significantly decreased in 3- and 5-year-old autotetraploid plants. Digital gene expression (DGE) analysis showed that the differentially expressed genes were mainly involved in IAA and BR pathways. microRNA390 was significantly upregulated according to microarray analysis. Exogenous application of IAA and BR promoted stem elongation of both apple plants grown in medium. The results show that dwarfing in autotetraploid apple plants is most likely regulated by IAA and BR. The dwarf phenotype of autotetraploid apple plants could be due to accumulation of miR390 after genome doubling, leading to upregulation of apple trans-acting short-interfering RNA 3 (MdTAS3) expression, which in turn downregulates the expression of MdARF3. Overall, this leads to partial interruption of the IAA and BR signal transduction pathway. Our study provides important insights into the molecular mechanisms underlying dwarfism in autopolyploid apple plants.

Immunophilin-like FKBP42/TWISTED DWARF1 Interacts with the Receptor Kinase BRI1 to Regulate Brassinosteroid Signaling in Arabidopsis

Mutation of the immunophilin-like FK506-binding protein TWISTED DWARF1 (FKBP42/TWD1) causes dwarf and twisted-organ phenotypes in Arabidopsis. However, the function of FKBP42 is not fully understood at the molecular level. Using genetic, physiological, and immunological experiments, we show here that FKBP42/TWD1 is necessary for brassinosteroid (BR) signal transduction. The twd1 mutant showed reduced BR sensitivity in growth responses and activation of the BZR1 transcription factor. However, twd1 showed normal responses to an inhibitor of BIN2/GSK3, suggesting that twd1 has a defect upstream of BIN2 in the BR signaling pathway. In vitro and in vivo assays showed that TWD1 interacts physically with the kinase domains of the BR receptor kinases BRI1 and BAK1. TWD1 is not required for normal localization of BRI1-GFP to the plasma membrane or for activation of the flagellin receptor kinase FLS2. Our results suggest that FKBP42/TWD1 plays a specific role in the activation of BRI1 receptor kinase.

TWISTED DWARF 1 Associates with BRASSINOSTEROID-INSENSITIVE 1 to Regulate Early Events of the Brassinosteroid Signaling Pathway

A genome-wide screen for mutants showing altered brassinosteroid (BR) sensitivity or bri1-like phenotypes resulted in the identification of two new mutant alleles of TWISTED DWARF 1 (TWD1), twd1-4, and twd1-5. Previous studies indicated that TWD1, also named as ULTRACURVATA 2 or FKBP42, associates with auxin efflux transporters and is essential for their biological functions. Although earlier reports showed that BR signaling is downregulated in twd1, how TWD1 is integrated in BR signaling has not been elucidated. Here, we provide genetic and biochemical evidence demonstrating that TWD1 interacts with the BR receptor BRI1 in vivo in a BR-independent manner. Further analyses indicated that TWD1 modulates the BR signal transduction not by altering ER quality control or protein abundance of BRI1; instead, TWD1 appears to be critical in BR-induced interaction of BRI1 and its co-receptor BAK1, as well as BR-induced phosphorylation of these two proteins. These results provide better understanding of the early events of the BR signaling pathway.

The molecular circuit of steroid signalling in plants.

Steroid hormones are key regulators of growth and physiology in both plants and animals. The plant steroid hormones known as brassinosteroids (BRs) are essential for a wide range of developmental processes throughout the life cycle. In contrast with animal steroid hormones, which act mostly through nuclear receptors, BRs act through a cell-surface receptor kinase. The BR signal transduction pathway from the cell-surface receptor to nuclear gene expression has been elucidated in great molecular detail, and thus serves as a paradigm for receptor kinase signalling in plants. Furthermore, several mechanisms of signal integration have been identified that explain how BRs and other hormonal and environmental signals co-regulate specific developmental outputs in a synergistic or antagonistic manner.

Multiple Interactions between Glucose and Brassinosteroid Signal Transduction Pathways in Arabidopsis Are Uncovered by Whole-Genome Transcriptional Profiling.

Brassinosteroid (BR) and glucose (Glc) regulate many common responses in plants. Here, we demonstrate that under etiolated growth conditions, extensive interdependence/overlap occurs between BR- and Glc-regulated gene expression as well as physiological responses. Glc could regulate the transcript level of 72% of BR-regulated genes at the whole-genome level, of which 58% of genes were affected synergistically and 42% of genes were regulated antagonistically. Presence of Glc along with BR in medium could affect BR induction/repression of 85% of BR-regulated genes. Glc could also regulate several genes involved in BR metabolism and signaling. Both BR and Glc coregulate a large number of genes involved in abiotic/biotic stress responses and growth and development. Physiologically, Glc and BR interact to regulate hypocotyl elongation growth of etiolated Arabidopsis (Arabidopsis thaliana) seedlings in a dose-dependent manner. Glc may interact with BR via a hexokinase1 (HXK1)-mediated pathway to regulate etiolated hypocotyl elongation. Brassinosteroid insensitive1 (BRI1) is epistatic to HXK1, as the Glc insensitive2bri1-6 double mutant displayed severe defects in hypocotyl elongation growth similar to its bri1-6 parent. Analysis of Glc and BR sensitivity in mutants defective in auxin response/signaling further suggested that Glc and BR signals may converge at S-phase kinase-associated protein1-Cullin-F-box-transport inhibitor response1/auxin-related f-box-auxin/indole-3-acetic acid-mediated auxin-signaling machinery to regulate etiolated hypocotyl elongation growth in Arabidopsis.

Brassinosteroid insensitive 1-associated kinase 1 (OsI-BAK1) is associated with grain filling and leaf development in rice

Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells.

Molecular characterization of BZR transcription factor family and abiotic stress induced expression profiling in Brassica rapa

BRASSINAZOLE-RESISTANT (BZR) transcription factors (TFs) are primarily well known as positive regulators of Brassinosteroid (BR) signal transduction in different plants. BR is a plant specific steroid hormone, which has multiple stress resistance functions besides various growth regulatory roles. Being an important regulator of the BR synthesis, BZR TFs might have stress resistance related activities. However, no stress resistance related functional study of BZR TFs has been reported in any crop plants so far. Therefore, this study identified 15 BZR TFs of Brassica rapa (BrBZR) from a genome-wide survey and characterized them through sequence analysis and expression profiling against several abiotic stresses. Various systematic in silico analysis of these TFs validated the fundamental properties of BZRs, where a high degree of similarity also observed with recognized BZRs of other plant species from the comparison studies. In the organ specific expression analyses, 6 BrBZR TFs constitutively expressed in flower developmental stages indicating their flower specific functions. Subsequently, from the stress resistance related expression profiles differential transcript abundance levels were observed by 6 and 11 BrBZRs against salt and drought stresses, respectively. All BrBZRs showed several folds up-regulation against exogenous ABA treatment. All BrBZRs also showed differential expression against low temperature stress treatments and these TFs were proposed as transcriptional activators of CBF cold response pathway of B. rapa. Notably, three BrBZRs gave co-responsive expression against all the stresses tested here, suggesting their multiple stress resistance related functions. Thus, the findings would be helpful in resolving the complex regulatory mechanism of BZRs in stress resistance and further functional genomics study of these potential TFs in different Brassica crops.

The molecular circuitry of brassinosteroid signaling.

Because they are tethered in space, plants have to make the most of their local growth environment. In order to grow in an ever-changing environment, plants constantly remodel their shapes. This adaptive attribute requires the orchestration of complex environmental signals at the cellular and organismal levels. A battery of small molecules, classically known as phytohormones, allows plants to change their body plan by using highly integrated signaling networks and transcriptional cascades. Amongst these hormones, brassinosteroids (BRs), the polyhydroxylated steroid of plants, influence plant responsiveness to the local environment and exquisitely promote, or interfere with, many aspects of plant development. The molecular circuits that wire steroid signals at the cell surface to the promoters of thousands of genes in the nucleus have been defined in the past decade. This review recapitulates how the transduction of BR signals impacts the temporally unfolding programs of plant growth. First, we summarize the paradigmatic BR signaling pathway acting primarily in cellular expansion. Secondly, we describe the current wiring diagram and the temporal dynamics of the BR signal transduction network. And finally we provide an overview of how key players in BR signaling act as molecular gates to transduce BR signals onto other signaling pathways.

Quantitative Proteomics Reveals the Role of Protein Phosphorylation in Rice Embryos during Early Stages of Germination

Seed germination begins with water uptake and ends with radicle emergence. A gel-free phosphoproteomic technique was used to investigate the role of protein phosphorylation events in the early stages of rice seed germination. Both seed weight and ATP content increased gradually during the first 24 h following imbibition. Proteomic analysis indicated that carbohydrate metabolism- and protein synthesis/degradation-related proteins were predominantly increased and displayed temporal patterns of expression. Analyses of cluster and protein-protein interactions indicated that the regulation of sucrose synthases and alpha-amylases was the central event controlling germination. Phosphoproteomic analysis identified several proteins involved in protein modification and transcriptional regulation that exhibited significantly temporal changes in phosphorylation levels during germination. Cluster analysis indicated that 12 protein modification-related proteins had a peak abundance of phosphoproteins at 12 h after imbibition. These results suggest that the first 12 h following imbibition is a potentially important signal transduction phase for the initiation of rice seed germination. Three core components involved in brassinosteroid signal transduction displayed significant increases in phosphoprotein abundance during the early stages of germination. Brassinolide treatment increased the rice seed germination rate but not the rate of embryonic axis elongation. These findings suggest that brassinosteroid signal transduction likely triggers seed germination.

Resolving the Brassinosteroids Signal Transduction Mechanisms by Single-Molecule Assays

Brassinosteroids (BRs) are the sixth class of plant hormones that involved in numerous plant development processes such as leaf expansion, shoot elongation and pollen tube formation. Once the signal transduction is initiated by the membrane receptor kinase BRI1 (brassinosteroid insensitive 1), the signal transmits from the cytoplasm to the nucleus and a number of genes will be regulated. The downstream signaling pathway is realized by three proteins: BIN2 (brassinosteroid insensitive 2), BES1 (BRI1 ems suppressor1) and a kind of 14-3-3s protein . BRs signaling pathway have been extensively studied via genetics, proteomics, genomics and cell biology techniques. However, these bulk methods can't follow the transduction process in situ or resolve molecular details at a rate matching the true signaling time-scale. Here we use a single molecular assay based on Total-Internally Reflected Fluorescence (TIRF) microscopy to observe the interaction of these three proteins. The result shows that BIN2 can phosphorylate BES1 on the order of seconds, and the dimeric 14-3-3s can only bind with BES1 in its phosphorylated form. In addition, we have, for the first time, found that the interaction between BIN2 and BES1 is oxygen dependent. This result may have implications on BRs signaling pathway's involvement of stress acclimation in plants.

Integration of Brassinosteroid Signal Transduction with the Transcription Network for Fiber Development and Drought Stress in Gossypium hirsutum L

Brassinosteroids (BRs) are a family of steroid hormones present ubiquitously in plant kingdoms and regulate wide range of physiological and developmental processes. BRs signal to regulate brassinosteroid transcription factors (BRTF’s), which regulate target genes for various BR responses. However, the role of BRs in developing fiber and during drought stress which is major limiting factor in its development is still limited. Our in silico modelling using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals that BRTF’s form a gene regulatory network (GRN) during cotton fiber development. Hierarchical clustering highlighted gene expression during acute dehydration response from early, peak and late elongation stage. SEA (Singular Enrichment Analysis) based gene ontology analysis demonstrate synergistic effect of BRs with other hormones like auxin. Our results provide a strong footprint of BRs based gene regulation in cotton fiber growth and drought stress.

Molecular evidence of the involvement of heat shock protein 90 in brassinosteroid signaling in Arabidopsis T87 cultured cells

A closer association of HSP90s with brassinosteroid signaling is suggested by the brassinosteroid-triggered formation of an HSP90-containing macromolecular complex and the direct interaction between HSP90.3 and BES1. Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that is reportedly involved in the proper folding, stabilization, intracellular trafficking, maintenance and degradation of numerous proteins, as well as the facilitation of cellular signaling in various organisms including plants. Brassinosteroids (BRs), a class of unique steroidal hormones, play crucial roles in plant growth and development. The interaction between HSP90 proteins and BR action has been poorly understood. Here, we present molecular evidence suggesting that HSP90 proteins have a function(s) in BR signal transduction. First, blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis linked immunoblotting demonstrated that a bioactive BR, brassinolide (BL), promotes the formation of some HSP90-containing macromolecular complexes with molecular weight more than 480 kDa in Arabidopsis T87 cultured cells. Second, HSP90.3, one of seven Arabidopsis HSP90 family proteins, was observed to interact in vitro with BRI1-EMS-SUPPRESSOR 1 (BES1), a transcription factor acting in BR signaling. Geldanamycin, an inhibitor of ATPase activity in HSP90, not only diminished HSP90.3 interaction with BES1 in vitro, but also suppressed BL-induced down-regulation of two BR biosynthesis genes, CONSTITUTIVE PHOTHOMORPHOGENESIS AND DWARFISM and DWARF4 in vivo. The results suggest the involvement of the HSP90/BES1 heterocomplexes in BR signaling-mediated feedback control in BR contents. Together, our results provide important clues to elucidate HSP90s' functions in the BR signaling pathway in Arabidopsis.

Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

Characterization and genetic mapping of a Photoperiod-sensitive dwarf 1 locus in rice (Oryza sativa L.)

Plant height is an important agronomic trait for crop architecture and yield. Most known factors determining plant height function in gibberellin or brassinosteroid biosynthesis or signal transduction. Here, we report a japonica rice (Oryza sativa ssp. japonica) dominant dwarf mutant, Photoperiod-sensitive dwarf 1 (Psd1). The Psd1 mutant showed impaired cell division and elongation, and a severe dwarf phenotype under long-day conditions, but nearly normal growth in short-day. The plant height of Psd1 mutant could not be rescued by gibberellin or brassinosteroid treatment. Genetic analysis with R1 and F2 populations determined that Psd1 phenotype was controlled by a single dominant locus. Linkage analysis with 101 tall F2 plants grown in a long-day season, which were derived from a cross between Psd1 and an indica cultivar, located Psd1 locus on chromosome 1. Further fine-mapping with 1017 tall F2 plants determined this locus on an 11.5-kb region. Sequencing analysis of this region detected a mutation site in a gene encoding a putative lipid transfer protein; the mutation produces a truncated C-terminus of the protein. This study establishes the genetic foundation for understanding the molecular mechanisms regulating plant cell division and elongation mediated by interaction between genetic and environmental factors.

Teaching an Old Hormone New Tricks: Cytosolic Ca2+ Elevation Involvement in Plant Brassinosteroid Signal Transduction Cascades

Brassinosteroids (BRs) are hormones that control many aspects of plant growth and development, acting at the cell level to promote division and expansion. BR regulation of plant and plant cell function occurs through altered expression of many genes. Transcriptional reprogramming downstream from cell perception of this hormone is currently known to be mediated by a phosphorylation/dephosphorylation ("phosphorelay") cascade that alters the stability of two master transcription regulators. Here, we provide evidence that BR perception by their receptor also causes an elevation in cytosolic Ca(2+), initiating a Ca(2+) signaling cascade in Arabidopsis (Arabidopsis thaliana) cell cytosol. BR-dependent increases in the expression of some genes (INDOLE-3-ACETIC ACID-INDUCIBLE1 and PHYTOCHROME B ACTIVATION-TAGGED SUPPRESSOR1) were impaired in wild-type plants by a Ca(2+) channel blocker and also in the defense-no-death (dnd1) mutant, which lacks a functional cyclic GMP-activated cell membrane Ca(2+)-conducting channel. Alternatively, mutations that impair the BR phosphorelay cascade did not much affect the BR-dependent expression of these genes. Similar effects of the Ca(2+) channel blocker and dnd1 mutation were observed on a BR plant growth phenotype, deetiolation of the seedling hypocotyl. Further evidence presented in this report suggests that a BR-dependent elevation in cyclic GMP may be involved in the Ca(2+) signaling cascade initiated by this hormone. The work presented here leads to a new model of the molecular steps that mediate some of the cell responses to this plant hormone.

Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling.

The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PM-located BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.

Stay-green plants: what do they tell us about the molecular mechanism of leaf senescence

A practical approach to increasing crop yields is to extend the duration of active photosynthesis. Stay-green is a term that is used to describe mutant and transgenic plants or cultivars with the trait of maintaining their leaves for a longer period of time than the wild-type or crosses from which they are derived. Analyzing stay-green genotypes contributes to our understanding of the molecular mechanism regulating leaf senescence which may allow us to extend the duration of active photosynthesis in crop plants. This article summarizes recent studies on stay-green plants and the insights they provide on the mechanism of leaf senescence. Briefly, mutations suppressing ethylene, abscisic acid, brassinosteroid, and strigolactone signal transduction or those activating cytokinin signaling often lead to stay-green phenotypes indicating a complex signaling network regulating leaf senescence. Developmentally regulated transcription factors, including NAC or WRKY family members, play key roles in the induction of leaf senescence and thus alteration in the activity of these transcription factors also result in stay-green phenotypes. Impairment in the enzymatic steps responsible for chlorophyll breakdown also leads to stay-green phenotypes. Some of these genotypes die in the middle of the process of chlorophyll breakdown due to the accumulation of toxic intermediates, while others appear to stay-green but their photosynthetic activity declines in a manner similar to wild-type plants. Alterations in certain metabolic pathways in chloroplasts (e.g., photosynthesis) can lead to a delayed onset of leaf senescence with maintenance of photosynthetic activity longer than wild-type plants, indicating that chloroplast metabolism can also affect the regulatory mechanism of leaf senescence.

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases.