This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol. In the assays described here, transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material using either thin-layer chromatography or phase-extraction. The second reporter system uses a kit to perform a simple wo-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with (125)I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.

Transformation of E. coli can be achieved using any of the four protocols in this unit. The first method using calcium chloride gives good transformation efficiencies, is simple to complete, requires no special equipment, and allows storage of competent cells. The alternate one-step method is considerably faster and also gives good transformation efficiencies (although they are somewhat lower). If considerably higher transformation efficiencies are needed, the third method using electroporation is simple, fast, and reliable. As in the calcium chloride protocol, prepared cells can be stored. The final method described is an adaptation of the electroporation protocol that allows direct transfer of vector DNA from yeast into E. coli.

BACKGROUND. The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants. RESULTS. We show that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation. We also show that this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants. CONCLUSIONS. The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. The data also suggest that the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.

Despite the recognition of H(2)O(2) as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H(2)O(2) signal is perceived and transduced in plant cells. We report here that H(2)O(2) is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H(2)O(2) can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H(2)O(2) effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture.

Sugar repression of photosynthetic genes is likely a central control mechanism mediating energy homeostasis in a wide range of algae and higher plants. It overrides light activation and is coupled to developmental and environmental regulations. How sugar signals are sensed and transduced to the nucleus remains unclear. To elucidate sugar-sensing mechanisms, we monitored the effects of a variety of sugars, glucose analogs, and metabolic intermediates on photosynthetic fusion genes in a sensitive and versatile maize protoplast transient expression system. The results show that sugars that are the substrates of hexokinase (HK) cause repression at a low concentration (1 to 10 mM), indicating a low degree of specificity and the irrelevance of osmotic change. Studies with various glucose analogs suggest that glucose transport across the plasma membrane is necessary but not sufficient to trigger repression, whereas subsequent phosphorylation by HK may be required. The effectiveness of 2-deoxyglucose, a nonmetabolizable glucose analog, and the ineffectiveness of various metabolic intermediates in eliciting repression eliminate the involvement of glycolysis and other metabolic pathways. Replenishing intracellular phosphate and ATP diminished by hexoses does not overcome repression. Because mannoheptulose, a specific HK inhibitor, blocks the severe repression triggered by 2-deoxyglucose and yet the phosphorylated products per se do not act as repression signals, we propose that HK may have dual functions and may act as a key sensor and signal transmitter of sugar repression in higher plants.

Using freshly isolated maize mesophyll protoplasts and a transient expression method, I showed that the transcriptional activity of seven maize photosynthetic gene promoters is specifically and coordinately repressed by the photosynthetic end products sucrose and glucose and by the exogenous carbon source acetate. Analysis of deleted, mutated, and hybrid promoters showed that sugars and acetate inhibit the activity of distinct positive upstream regulatory elements without a common consensus. The metabolic repression of photosynthetic genes overrides other forms of regulation, e.g., light, tissue type, and developmental stage. Repression by sugars and repression by acetate are mediated by different mechanisms. The identification of conditions that avoid sugar repression overcomes a major obstacle to the study of photosynthetic gene regulation in higher plants.

The mechanisms by which higher plants recognize and respond to sugars are largely unknown. Here, we present evidence that the first enzyme in the hexose assimilation pathway, hexokinase (HXK), acts as a sensor for plant sugar responses. Transgenic Arabidopsis plants expressing antisense hexokinase (AtHXK) genes are sugar hyposensitive, whereas plants overexpressing AtHXK are sugar hypersensitive. The transgenic plants exhibited a wide spectrum of altered sugar responses in seedling development and in gene activation and repression. Furthermore, overexpressing the yeast sugar sensor YHXK2 caused a dominant negative effect by elevating HXK catalytic activity but reducing sugar sensitivity in transgenic plants. The result suggests that HXK is a dual-function enzyme with a distinct regulatory function not interchangeable between plants and yeast.

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.

Stress responses in plants involve changes in the transcription of specific genes. The constitutively active mutants of two related Ca2+-dependent protein kinases (CDPK1 and CDPK1a) activate a stress-inducible promoter, bypassing stress signals. Six other plant protein kinases, including two distinct CDPKs, fail to mimic this stress signaling. The activation is abolished by a CDPK1 mutation in the kinase domain and diminished by a constitutively active protein phosphatase 2C that is capable of blocking responses to the stress hormone abscisic acid. A variety of functions are mediated by different CDPKs. CDPK1 and CDPK1a may be positive regulators controlling stress signal transduction in plants.

Glucose is an essential signaling molecule that controls plant development and gene expression through largely unknown mechanisms. To initiate the dissection of the glucose signal transduction pathway in plants by using a genetic approach, we have identified an Arabidopsis mutant, gin1 (glucose-insensitive), in which glucose repression of cotyledon greening and expansion, shoot development, floral transition, and gene expression is impaired. Genetic analysis indicates that GIN1 acts downstream of the sensor hexokinase in the glucose signaling pathway. Surprisingly, gin1 insensitivity to glucose repression of cotyledon and shoot development is phenocopied by ethylene precursor treatment of wild-type plants or by constitutive ethylene biosynthesis and constitutive ethylene signaling mutants. In contrast, the ethylene insensitive mutant etr1-1 exhibits glucose hypersensitivity. Epistasis analysis places GIN1 downstream of the ethylene receptor, ETR1, and defines a new branch of ethylene signaling pathway that is uncoupled from the triple response induced by ethylene. The isolation and characterization of gin1 reveal an unexpected convergence between the glucose and the ethylene signal transduction pathways. GIN1 may function to balance the control of plant development in response to metabolic and hormonal stimuli that act antagonistically.

The green-fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat-shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single-cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co-factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence-activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co-transfection, transformation, protein trafficking and localization, protein-protein interaction, cell separation and purification, and cell lineage in higher plants.

Sugars have signaling roles in a wide variety of developmental processes in plants. To elucidate the regulatory components that constitute the glucose signaling network governing plant growth and development, we have isolated and characterized two Arabidopsis glucose insensitive mutants, gin5 and gin6, based on a glucose-induced developmental arrest during early seedling morphogenesis. The T-DNA-tagged gin6 mutant abrogates the glucose-induced expression of a putative transcription factor, ABI4, previously shown to be involved in seed-specific abscisic acid (ABA) responses. Thus, ABI4 might be a regulator involved in both glucose- and seed-specific ABA signaling. The characterization of the gin5 mutant, on the other hand, reveals that glucose-specific accumulation of ABA is essential for hexokinase-mediated glucose responses. Consistent with this result, we show that three ABA-deficient mutants (aba1-1, aba2-1, and aba3-2) are also glucose insensitive. Exogenous ABA can restore normal glucose responses in gin5 and aba mutants but not in gin6 plants. Surprisingly, only abi4 and abi5-1 but not other ABA-insensitive signaling mutants (abi1-1, abi2-1, and abi3-1) exhibit glucose insensitivity, indicating the involvement of a distinct ABA signaling pathway in glucose responses. These results provide the first direct evidence to support a novel and central role of ABA in plant glucose responses mediated through glucose regulation of both ABA levels by GIN5 and ABA signaling by GIN6/ABI4.