Claire E. Hutchison

The Arabidopsis Histidine Phosphotransfer Proteins Are Redundant Positive Regulators of Cytokinin Signaling

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.

Cytokinin signaling in Arabidopsis

Cytokinins have been implicated in many developmental processes and environmental responses of plants, including leaf senescence, apical dominance, chloroplast development, anthocyanin production, and the regulation of cell division and sink/source relationships. They were identified in the

Cytokinin Regulates Type-A Arabidopsis Response Regulator Activity and Protein Stability via Two-Component Phosphorelay

The plant hormone cytokinin regulates many aspects of growth and development. Cytokinin signaling involves His kinase receptors that perceive cytokinin and transmit the signal via a multistep phosphorelay similar to bacterial two-component signaling systems. The final targets of this phosphorelay are a set of Arabidopsis thaliana Response Regulator (ARR) proteins containing a receiver domain with a conserved Asp phosphorylation site. One class of these, the type-A ARRs, are negative regulators of cytokinin signaling that are rapidly transcriptionally upregulated in response to cytokinin. In this study, we tested the role of phosphorylation in type-A ARR function. Our results indicate that phosphorylation of the receiver domain is required for type-A ARR function and suggest that negative regulation of cytokinin signaling by the type-A ARRs most likely involves phosphorylation-dependent interactions. Furthermore, we show that a subset of the type-A ARR proteins are stabilized in response to cytokinin in part via phosphorylation. These studies shed light on the mechanism by which type-A ARRs act to negatively regulate cytokinin signaling and reveal a novel mechanism by which cytokinin controls type-A ARR function.

Two-Component Elements Mediate Interactions between Cytokinin and Salicylic Acid in Plant Immunity

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant–pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant–pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA–dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA–dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens.

The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

CDKN2B expression in adipose tissue of familial combined hyperlipidemia patients

The purpose of this study was to determine the core biological processes perturbed in the subcutaneous adipose tissue of familial combined hyperlipidemia (FCHL) patients. Annotation of FCHL and control microarray datasets revealed a distinctive FCHL transcriptome, characterized by gene expression changes regulating five overlapping systems: the cytoskeleton, cell adhesion and extracellular matrix; vesicular trafficking; lipid homeostasis; and cell cycle and apoptosis. Expression values for the cell-cycle inhibitor CDKN2B were increased, replicating data from an independent FCHL cohort. In 3T3-L1 cells, CDKN2B knockdown induced C/EBPα expression and lipid accumulation. The minor allele at SNP site rs1063192 (C) was predicted to create a perfect seed for the human miRNA-323b-5p. A miR-323b-5p mimic significantly reduced endogenous CDKN2B protein levels and the activity of a CDKN2B 3′UTR luciferase reporter carrying the rs1063192 C allele. Although the allele displayed suggestive evidence of association with reduced CDKN2B mRNA in the MuTHER adipose tissue dataset, family studies suggest the association between increased CDKN2B expression and FCHL-lipid abnormalities is driven by factors external to this gene locus. In conclusion, from a comparative annotation analysis of two separate FCHL adipose tissue transcriptomes and a subsequent focus on CDKN2B, we propose that dysfunctional adipogenesis forms an integral part of FCHL pathogenesis.

The Endoplasmic Reticulum Coat Protein II Transport Machinery Coordinates Cellular Lipid Secretion and Cholesterol Biosynthesis

Background: Sar1 mediates the onward transport of ER cargo. Results: Sar1B promotes VLDL secretion, whereas Sar1A antagonizes this activity, and a deficit of both reduces cholesterol biosynthesis. Conclusion: Sar1B independently of and through its lipoprotein secretion function promotes the expression of genes regulating cholesterol biosynthesis. Significance: Sar1B-mediated transport activities contribute to both the functional integrity of the ER membrane and blood cholesterol levels. Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1Bs repertoire of transport functions.

Signaling via Histidine-Containing Phosphotransfer Proteins in Arabidopsis.

The Arabidopsis genome encodes a number of proteins with similarity to two-component phosphorelay signaling elements, including hybrid receptor histidine kinases, two classes of response regulator proteins (type-A and type-B ARRs) and a family of six histidine-containing phosphotransfer proteins (AHPs), five of which contain a conserved His residue that is required for phosphorelay signaling. The current model for cytokinin signaling includes a multistep phosphorelay: three histidine kinases and at least five type-B ARRs have been shown to act as positive regulators of cytokinin signaling, while a number of type-A ARRs, and AHP6, act as negative regulators of the pathway. In our recent Plant Cell paper, we provided genetic evidence that at least four AHPs can act as positive regulators of cytokinin signaling, affecting responses to cytokinin in the root and the shoot. In this addendum, we discuss the role of AHPs in cytokinin signaling and speculate on their potential interactions with other signaling pathways in Arabidopsis.

A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene

Abstract Previous studies indicate that the ability of Arabidopsis seedlings to recover normal growth following an ethylene treatment involves histidine kinase activity of the ethylene receptors. As histidine kinases can function as inputs for a two‐component signaling system, we examined loss‐of‐function mutants involving two‐component signaling elements. We find that mutants of phosphotransfer proteins and type‐B response regulators exhibit a defect in their ethylene growth recovery response similar to that found with the loss‐of‐function ethylene receptor mutant etr1‐7. The ability of two‐component signaling elements to regulate the growth recovery response to ethylene functions independently from their well‐characterized role in cytokinin signaling, based on the analysis of cytokinin receptor mutants as well as following chemical inhibition of cytokinin biosynthesis. Histidine kinase activity of the receptor ETR1 also facilitates growth recovery in the ethylene hypersensitive response, which is characterized by a transient decrease in growth rate when seedlings are treated continuously with a low dose of ethylene; however, this response was found to operate independently of the type‐B response regulators. These results indicate that histidine kinase activity of the ethylene receptor ETR1 performs two independent functions: (a) regulating the growth recovery to ethylene through a two‐component signaling system involving phosphotransfer proteins and type‐B response regulators and (b) regulating the hypersensitive response to ethylene in a type‐B response regulator independent manner.