Biomedical subjects
Publications and source records attributed to J Sheen.
Plant sugar signalling operates in a complex network with plant-specific hormone signalling pathways. Hexokinase was identified as an evolutionarily conserved glucose sensor that integrates light, hormone and nutrient signalling to control plant growth and development.
E896 has measured Lambda production in 11.6A GeV/c Au-Au collisions over virtually the whole rapidity phase space. The midrapidity p(t) distributions have been measured for the first time at this energy and appear to indicate that the Lambda hyperons have different freeze-out conditions than protons. A comparison with the relativistic quantum molecular dynamics model shows that while there is good shape agreement at high rapidity the model predicts significantly different slopes of the m(t) spectra at midrapidity. The data, where overlap occurs, are consistent with previously reported measurements.
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.
Phenylalanine ammonia-lyase (PAL) is a key enzyme in pathogen defence, stress response and secondary metabolism and is subject to post-translational phosphorylation. In order to address the significance of this phenomenon it is necessary to identify the protein kinase (PK) responsible and place it in its regulatory circuit. Using protoplast transient expression of Arabidopsis kinase genes coupled to immunocomplex kinase assay, it has been possible to screen for specific PAL kinase. We show here that AtCPK1 (calcium dependent PK), but not other closely related PKs could phosphorylate both a recombinant PAL protein and a peptide (SRVAKTRTLTTA) that is a site phosphorylated in vivo. Identification of the specific CDPK as a PAL kinase now opens up the possibility of exploring the calcium link in biotic stress signalling, salicylate and phytoalexin production as well as the significance of PAL phosphorylation. The protoplast transient expression system is a potentially powerful method to determine and screen for plant gene functions utilising genomic and proteomic data.
Mitogen-activated protein kinase (MAPK) cascades have emerged as a universal signal transduction mechanism that connects diverse receptors/sensors to cellular and nuclear responses in eukaryotes. Recent studies in plants indicate that MAPK cascades are vital to fundamental physiological functions involved in hormonal responses, cell cycle regulation, abiotic stress signaling, and defense mechanisms. New findings have revealed the complexity and redundancy of the signaling components, the antagonistic nature of distinct pathways, and the use of both positive and negative regulatory mechanisms.
To play an essential role in C4 photosynthesis, the maize C4 phosphoenolpyruvate carboxylase gene (PPCZm1) acquired many new expression features, such as leaf specificity, mesophyll specificity, light inducibility and high activity, that distinguish the unique C4 PPC from numerous non-C4 PPC genes in maize. We present here the first investigation of the developmental, cell-specific, light and metabolic regulation of the homologous C4 PPCZm1 promoter in stable transgenic maize plants. We demonstrate that the 1.7 kb of the 5'-flanking region of the PPCZm1 gene is sufficient to direct the C4-specific expression patterns of beta-glucuronidase (GUS) activity, as a reporter, in stable transformed maize plants. In light-grown shoots, GUS expression was strongest in all developing and mature mesophyll cells in the leaf, collar and sheath. GUS activity was also detected in mesophyll cells in the outer husks of ear shoots and in the outer glumes of staminate spikelets. We did not observe histological localization of GUS activity in light- or dark-grown callus, roots, silk, developing or mature kernels, the shoot apex, prop roots, or pollen. In addition, we used the stable expressing transformants to conduct and quantify physiological induction studies. Our results indicate that the expression of the C4 PPCZm1-GUS fusion gene is mesophyll-specific and influenced by development, light, glucose, acetate and chloroplast biogenesis in transgenic maize plants. These studies suggest that the adoption of DNA regulatory elements for C4-specific gene expression is a crucial step in C4 gene evolution.
Abscisic acid (ABA) induces genes that are highly expressed during late embryogenesis, but suppresses gibberellin (GA)-responsive genes essential for seed germination and seedling growth. Promoter elements necessary and sufficient for ABA up- and down-regulation of gene expression have been previously defined in barley aleurone layers. We have studied the effect of a protein phosphatase 2C, ABI1, an ABA-inducible protein kinase, PKABA1, and a transcription factor, VP1, on ABA action in a barley aleurone transient expression system. The observations have allowed us to dissect ABA signal transduction pathways leading to either induction or suppression of gene expression. The ABA induction of embryogenesis genes is highly inhibited in the presence of a mutated protein phosphatase 2C, encoded by the abi1-1 dominant mutant gene that is known to block ABA responses in Arabidopsis. However, the abi1-1 gene product has no effect on the ABA suppression of a GA-responsive alpha-amylase gene. On the other hand, PKABA1 suppresses the expression of alpha-amylase genes, but has little effect on ABA up-regulated genes. Therefore, it appears that ABA induction and suppression follow two separate signal transduction pathways with the former inhibited by ABI1 and the latter modulated by PKABA1. The presence of VP1 enhances the ABA induction of late embryogenesis genes, but also suppresses germination specific genes. A schematic model based on these observations is presented to explain the effect of these regulatory proteins on ABA-mediated gene expression.
Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.
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.
Here we report a novel Arabidopsis dwarf mutant, fackel-J79, whose adult morphology resembles that of brassinosteroid-deficient mutants but also displays distorted embryos, supernumerary cotyledons, multiple shoot meristems, and stunted roots. We cloned the FACKEL gene and found that it encodes a protein with sequence similarity to both the human sterol reductase family and yeast C-14 sterol reductase and is preferentially expressed in actively growing cells. Biochemical analysis indicates that the fk-J79 mutation results in deficient C-14 sterol reductase activity, abnormal sterol composition, and reduction of brassinosteroids (BRs). Unlike other BR-deficient mutants, the defect of hypocotyl elongation in fk-J79 cannot be corrected by exogenous BRs. The unique phenotypes and sterol composition in fk-J79 indicate crucial roles of sterol regulation and signaling in cell division and cell expansion in embryonic and post-embryonic development in plants.
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.
Previous studies have revealed a central role of Arabidopsis thaliana hexokinases (AtHXK1 and AtHXK2) in the glucose repression of photosynthetic genes and early seedling development. However, it remains unclear whether HXK can modulate the expression of diverse sugar-regulated genes. On the basis of the results of analyses of gene expression in HXK transgenic plants, we suggest that three distinct glucose signal transduction pathways exist in plants. The first is an AtHXK1-dependent pathway in which gene expression is correlated with the AtHXK1-mediated signaling function. The second is a glycolysis-dependent pathway that is influenced by the catalytic activity of both AtHXK1 and the heterologous yeast Hxk2. The last is an AtHXK1-independent pathway in which gene expression is independent of AtHXK1. Further investigation of HXK transgenic Arabidopsis discloses a role of HXK in glucose-dependent growth and senescence. In the absence of exogenous glucose, plant growth is limited to the seedling stage with restricted true leaf development even after a 3-week culture on MS medium. In the presence of glucose, however, over-expressing Arabidopsis or yeast HXK in plants results in the repression of growth and true leaf development, and early senescence, while under-expressing AtHXK1 delays the senescence process. These studies reveal multiple glucose signal transduction pathways that control diverse genes and processes that are intimately linked to developmental stages and environmental conditions.
We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.
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Recent studies indicate that, in a manner similar to classical plant hormones, sugars can act as signaling molecules that control gene expression and developmental processes in plants. Crucial evidence includes uncoupling glucose signaling from its metabolism, identification of glucose sensors, and isolation and characterization of mutants and other regulatory components in plant sugar signal transduction pathways. The emerging scenario points to the existence of a complex signaling network that interconnects transduction pathways from sugars and other hormone and nutrient signals.
We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.
The plant hormone auxin activates many early response genes that are thought to be responsible for diverse aspects of plant growth and development. It has been proposed that auxin signal transduction is mediated by a conserved signalling cascade consisting of three protein kinases: the mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). Here we show that a specific plant MAPKKK, NPK1, activates a MAPK cascade that leads to the suppression of early auxin response gene transcription. A mutation in the kinase domain abolishes NPK1 activity, and the presence of the carboxy-terminal domain diminishes the kinase activity. Moreover, the effects of NPK1 on the activation of a MAPK and the repression of early auxin response gene transcription are specifically eliminated by a MAPK phosphatase. Transgenic tobacco plants overexpressing the NPK1 kinase domain produced seeds defective in embryo and endosperm development. These results suggest that auxin sensitivity may be balanced by antagonistic signalling pathways that use a distinct MAPK cascade in higher 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.