Ildoo Hwang

Two-component circuitry in Arabidopsis cytokinin signal transduction

Cytokinins are essential plant hormones that are involved in shoot meristem and leaf formation, cell division, chloroplast biogenesis and senescence. Although hybrid histidine protein kinases have been implicated in cytokinin perception in Arabidopsis, the action of histidine protein kinase receptors and the downstream signalling pathway has not been elucidated to date. Here we identify a eukaryotic two-component signalling circuit that initiates cytokinin signalling through distinct hybrid histidine protein kinase activities at the plasma membrane. Histidine phosphotransmitters act as signalling shuttles between the cytoplasm and nucleus in a cytokinin-dependent manner. The short signalling circuit reaches the nuclear target genes by enabling nuclear response regulators ARR1, ARR2 and ARR10 as transcription activators. The cytokinin-inducible ARR4, ARR5, ARR6 and ARR7 genes encode transcription repressors that mediate a negative feedback loop in cytokinin signalling. Ectopic expression in transgenic Arabidopsis of ARR2, the rate-limiting factor in the response to cytokinin, is sufficient to mimic cytokinin in promoting shoot meristem proliferation and leaf differentiation, and in delaying leaf senescence.

Activation of Glucosidase via Stress-Induced Polymerization Rapidly Increases Active Pools of Abscisic Acid

Abscisic acid (ABA) is a phytohormone critical for plant growth, development, and adaptation to various stress conditions. Plants have to adjust ABA levels constantly to respond to changing physiological and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. Here we report that AtBG1, a beta-glucosidase, hydrolyzes glucose-conjugated, biologically inactive ABA to produce active ABA. Loss of AtBG1 causes defective stomatal movement, early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas plants with ectopic AtBG1 accumulate higher ABA levels and display enhanced tolerance to abiotic stress. Dehydration rapidly induces polymerization of AtBG1, resulting in a 4-fold increase in enzymatic activity. Furthermore, diurnal increases in ABA levels are attributable to polymerization-mediated AtBG1 activation. We propose that the activation of inactive ABA pools by polymerized AtBG1 is a mechanism by which plants rapidly adjust ABA levels and respond to changing environmental cues.

Two-Component Signal Transduction Pathways in Arabidopsis

The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.

Cytokinin Signaling Networks

Despite long-standing observations on diverse cytokinin actions, the discovery path to cytokinin signaling mechanisms was tortuous. Unyielding to conventional genetic screens, experimental innovations were paramount in unraveling the core cytokinin signaling circuitry, which employs a large repertoire of genes with overlapping and specific functions. The canonical two-component transcription circuitry involves His kinases that perceive cytokinin and initiate signaling, as well as His-to-Asp phosphorelay proteins that transfer phosphoryl groups to response regulators, transcriptional activators, or repressors. Recent advances have revealed the complex physiological functions of cytokinins, including interactions with auxin and other signal transduction pathways. This review begins by outlining the historical path to cytokinin discovery and then elucidates the diverse cytokinin functions and key signaling components. Highlights focus on the integration of cytokinin signaling components into regulatory networks in specific contexts, ranging from molecular, cellular, and developmental regulations in the embryo, root apical meristem, shoot apical meristem, stem and root vasculature, and nodule organogenesis to organismal responses underlying immunity, stress tolerance, and senescence.

The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis

Cytokinins affect plant immunity to various pathogens; however, the mechanisms coupling plant-derived cytokinins to pathogen responses have been elusive. Here, we found that plant-derived cytokinins promote resistance of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst). Modulated cytokinin levels or signaling activity in CKX- or IPT-overexpressing plants or in ahk2 ahk3 mutants correlated with altered resistance. In fact, the cytokinin-activated transcription factor ARR2 contributes specifically to Pst resistance. The salicylic acid (SA) response factor TGA3 binds ARR2, and mutation of TGA-binding cis-elements in the Pr1 promoter abolished cytokinin- and ARR2-dependent Pr1 activation. Cytokinin treatment did not increase pathogen resistance in tga3 plants, as the cytokinin-dependent induction of Pr1 was eliminated. Moreover, SA signaling enhanced binding of ARR2/TGA3 to the Pr1 promoter. Taken together, these results show that cytokinins modulate the SA signaling to augment resistance against Pst, a process in which the interaction between TGA3 and ARR2 is important.

Nucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential in Arabidopsis Brassinosteroid Signaling

Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating the phosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1 (BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and a GSK3 kinase, BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown how the perception of BR at the plasma membrane connects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmic shuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3 proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for 14-3-3 binding, another phosphorylation domain in BZR1 is required for the BIN2-induced nuclear export of BZR1. Mutations of putative phosphorylation sites in two distinct domains enhance the nuclear retention of BZR1 and BR responses in transgenic plants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

The response regulator 2 mediates ethylene signalling and hormone signal integration in Arabidopsis

Hormones are important regulators of plant growth and development. In Arabidopsis, perception of the phytohormones ethylene and cytokinin is accomplished by a family of sensor histidine kinases including ethylene‐resistant (ETR) 1 and cytokinin‐response (CRE) 1. We identified the Arabidopsis response regulator 2 (ARR2) as a signalling component functioning downstream of ETR1 in ethylene signal transduction. Analyses of loss‐of‐function and ARR2‐overexpressing lines as well as functional assays in protoplasts indicate an important role of ARR2 in mediating ethylene responses. Additional investigations indicate that an ETR1‐initiated phosphorelay regulates the transcription factor activity of ARR2. This mechanism may create a novel signal transfer from endoplasmic reticulum‐associated ETR1 to the nucleus for the regulation of ethylene‐response genes. Furthermore, global expression profiling revealed a complex ARR2‐involving two‐component network that interferes with a multitude of different signalling pathways and thereby contributes to the highly integrated signal processing machinery in higher plants.

Control of plant germline proliferation by SCF FBL17 degradation of cell cycle inhibitors

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1–Cullin1–F-box protein (SCF) E3 ubiquitin ligase complex (SCFFBL17) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCFFBL17 as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.

Cytokinins and plant immunity: old foes or new friends?

Cytokinins are plant growth promoting hormones involved in the specification of embryonic cells, maintenance of meristematic cells, shoot formation and development of vasculature. Cytokinins have also emerged as a major factor in plant-microbe interactions during nodule organogenesis and pathogenesis. Microbe-originated cytokinins confer abnormal hypersensitivity of cytokinins to plants, augmenting the sink activity of infected regions. However, recent findings have shed light on a distinct role of cytokinins in plant immune responses. Plant-borne cytokinins systemically induce resistance against pathogen infection. This resistance is orchestrated by endogenous cytokinin and salicylic acid signaling. Here, we discuss how plant- and pathogen-derived cytokinins inversely affect the plant defense response. In addition, we consider the molecular mechanisms underlying plant-derived cytokinin action in plant immunity.

Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.