The protoplast transient expression system has become a powerful and popular tool for studying molecular mechanisms underlying various plant signal transduction pathways. Arabidopsis mesophyll protoplasts display intact and active physiological responses and are easy to isolate and transfect, which facilitate high-throughput screening and systematic and genome-wide characterization of gene functions. The system is suitable for most Arabidopsis accessions and mutant plants. Genetic complementation of mutant defective in sensor functions, gene expression, enzymatic activities, protein interactions, and protein trafficking can be easily designed and explored in cell-based assays. Here, we describe the detailed protocols for protoplast isolation, polyethylene glycol-calcium transfection, and different assays for quantifying the output of various signaling pathways.

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.

There is remarkable conservation in the recognition of pathogen-associated molecular patterns (PAMPs) by innate immune responses of plants, insects and mammals. We developed an Arabidopsis thaliana leaf cell system based on the induction of early-defence gene transcription by flagellin, a highly conserved component of bacterial flagella that functions as a PAMP in plants and mammals. Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors that function downstream of the flagellin receptor FLS2, a leucine-rich-repeat (LRR) receptor kinase. Activation of this MAPK cascade confers resistance to both bacterial and fungal pathogens, suggesting that signalling events initiated by diverse pathogens converge into a conserved MAPK cascade.

Glucose modulates many vital processes in photosynthetic plants. Analyses of Arabidopsis glucose insensitive2 (gin2) mutants define the physiological functions of a specific hexokinase (HXK1) in the plant glucose-signaling network. HXK1 coordinates intrinsic signals with extrinsic light intensity. HXK1 mutants lacking catalytic activity still support various signaling functions in gene expression, cell proliferation, root and inflorescence growth, and leaf expansion and senescence, thus demonstrating the uncoupling of glucose signaling from glucose metabolism. The gin2 mutants are also insensitive to auxin and hypersensitive to cytokinin. Plants use HXK as a glucose sensor to interrelate nutrient, light, and hormone signaling networks for controlling growth and development in response to the changing environment.

Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging.

The transient gene expression system using Arabidopsis mesophyll protoplasts has proven an important and versatile tool for conducting cell-based experiments using molecular, cellular, biochemical, genetic, genomic and proteomic approaches to analyze the functions of diverse signaling pathways and cellular machineries. A well-established protocol that has been extensively tested and applied in numerous experiments is presented here. The method includes protoplast isolation, PEG-calcium transfection of plasmid DNA and protoplast culture. Physiological responses and high-throughput capability enable facile and cost-effective explorations as well as hypothesis-driven tests. The protoplast isolation and DNA transfection procedures take 6-8 h, and the results can be obtained in 2-24 h. The cell system offers reliable guidelines for further comprehensive analysis of complex regulatory mechanisms in whole-plant physiology, immunity, growth and development.

Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA.

In plants, numerous Ca(2+)-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.

Glucose is a global regulator of growth and metabolism that is evolutionarily conserved from unicellular microorganisms to multicellular animals and plants. In photosynthetic plants, glucose shows hormone-like activities and modulates many essential processes, including embryogenesis, germination, seedling development, vegetative growth, reproduction and senescence. Genetic and phenotypic analyses of Arabidopsis mutants with glucose-insensitive (gin) and glucose-oversensitive (glo) phenotypes have identified an unexpected antagonistic interaction between glucose and the plant stress hormone ethylene. The ethylene-insensitive etr1 and ein2 mutants have glo phenotypes, whereas the constitutive ethylene signalling mutant ctr1 is allelic to gin4 (refs 4, 5). The precise molecular mechanisms underlying the complex signalling network that governs plant growth and development in response to nutrients and plant hormones are mostly unknown. Here we show that glucose enhances the degradation of ETHYLENE-INSENSITIVE3 (EIN3), a key transcriptional regulator in ethylene signalling, through the plant glucose sensor hexokinase. Ethylene, by contrast, enhances the stability of EIN3. The ein3 mutant has a glo phenotype, and overexpression of EIN3 in transgenic Arabidopsis decreases glucose sensitivity.

Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabeled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways using different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging. Expected online publication date for the Annual Review of Plant Biology Volume 57 is April 28, 2006. Please see http://www.annualreviews.org/catalog/pub_dates.asp for revised estimates.

Sugars modulate many vital processes that are also controlled by hormones during plant growth and development. Characterization of sugar-signalling mutants in Arabidopsis has unravelled a complex signalling network that links sugar responses to two plant stress hormones--abscisic acid and ethylene--in opposite ways. Recent molecular analyses have revealed direct, extensive glucose control of abscisic acid biosynthesis and signalling genes that partially antagonizes ethylene signalling during seedling development under light. Glucose and abscisic acid promote growth at low concentrations but act synergistically to inhibit growth at high concentrations. The effects of sugar and osmotic stress on morphogenesis and gene expression are distinct. The plasticity of plant growth and development are exemplified by the complex interplay of sugar and hormone signalling.