Gillian A. Nimmo

Alternative Splicing Mediates Responses of the Arabidopsis Circadian Clock to Temperature Changes

The circadian clock is a timing device that allows plants to anticipate environmental changes rather than just respond to them. This work demonstrates that alternative splicing of clock gene transcripts is one of the mechanisms that regulate the clock, particularly in response to changes in temperature. Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes LATE ELONGATED HYPOCOTYL (LHY) and PSEUDO RESPONSE REGULATOR7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.

The Circadian Clock in Arabidopsis Roots Is a Simplified Slave Version of the Clock in Shoots

The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.

BHLH32 modulates several biochemical and morphological processes that respond to Pi starvation in Arabidopsis

P(i) (inorganic phosphate) limitation severely impairs plant growth and reduces crop yield. Hence plants have evolved several biochemical and morphological responses to P(i) starvation that both enhance uptake and conserve use. The mechanisms involved in P(i) sensing and signal transduction are not completely understood. In the present study we report that a previously uncharacterized transcription factor, BHLH32, acts as a negative regulator of a range of P(i) starvation-induced processes in Arabidopsis. In bhlh32 mutant plants in P(i)-sufficient conditions, expression of several P(i) starvation-induced genes, formation of anthocyanins, total P(i) content and root hair formation were all significantly increased compared with the wild-type. Among the genes negatively regulated by BHLH32 are those encoding PPCK (phosphoenolpyruvate carboxylase kinase), which is involved in modifying metabolism so that P(i) is spared. The present study has shown that PPCK genes are rapidly induced by P(i) starvation leading to increased phosphorylation of phosphoenolpyruvate carboxylase. Furthermore, several Arabidopsis proteins that regulate epidermal cell differentiation [TTG1 (TRANSPARENT TESTA GLABRA1), GL3 (GLABRA3) and EGL3 (ENHANCER OF GL3)] positively regulate PPCK gene expression in response to P(i) starvation. BHLH32 can physically interact with TTG1 and GL3. We propose that BHLH32 interferes with the function of TTG1-containing complexes and thereby affects several biochemical and morphological processes that respond to P(i) availability.

Changes in the kinetic properties and phosphorylation state of phosphoenolpyruvate carboxylase in Zea mays leaves in reponse to light and dark

In plants such as Zea mays that carry out C4 metabolism, phosphoenolpyruvate carboxylase catalyses the primary fixation of atmospheric CO2. The properties of this enzyme from Z. mays leaves kept in light and in darkness are different. In brightly illuminated leaves, which are actively fixing CO2, the enzyme is less sensitive to feedback inhibition by malate and is phosphorylated on one or more serine residues. In darkened leaves, which are not photosynthesising, the enzyme is more sensitive to inhibition by malate and is much less phosphorylated. This indicates that the activity of the enzyme is controlled by a reversible phosphorylation.

Diurnal changes in the properties of phosphoenolpyruvate carboxylase in Bryophyllum leaves: a possible co valent modification

In plants that show Crassulacean acid metabolism, phosphoenolpyruvate carboxylase catalyses the key step of CO2 fixation at night. We show here that the properties of this enzyme from Bryophyllum fedtschenkoi undergo marked changes between night and day; the night form is much less sensitive to feedback inhibition by malate than is the day form. Incubation of leaves with 32Pi followed by extraction and immunoprecipitation of phosphoenolpyruvate carboxylase showed that only the night form contained 32P. This suggests that the activity of the enzyme is controlled by a covalent modification mechanism.

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO2-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO2 and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO2 was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO2 uptake did not correlate in phase or period.

[40] Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle

Publisher Summary Protein Phosphatase inhibitor-I becomes inhibitory toward protein phosphatase-I only when it has been phosphorylated by cAMPdependent protein kinase, and is likely to play an important role in the control of glycogen metabolism by epinephrine in skeletal muscle. Inhibitor- 2 is a subunit of the cytosolic form of protein phosphatase-l, termed phosphatase-1 1 . Inhibitor-1 and inhibitor-2 are assayed by their ability to inhibit the dephosphorylation of phosphorylase a catalyzed by the free catalytic subtunit of protein phosphatase-1. They are effective at nanomolar levels, which is similar to the concentration of protein phosphatase-1 in the assays. Consequently, the extent of inhibition decreases as the concentration of protein phosphatase-1 increases and vice versa. It is, therefore, important to standardize the protein phosphatase concentration in the assays. Inhibitor-1 is in its dephosphorylated form at each stage of the preparation and must, therefore, be phosphorylated prior to assay. Inhibitor-2 is also diluted in solution A + 0.03% (w/v) Brij-35 and assayed in an identical manner to inhibitor-l, except that prior phosphorylation with cAMP-dependent protein kinase is omitted, and preincubation with protein phosphatase-1 is for 10 rather than 2 min, before addition of 32 p-labeled phosphorylase a .

THE DISTRIBUTION OF SOLUBLE AND MEMBRANE‐BOUND FORMS OF GLUTAMINASE IN PIG BRAIN

Abstract— About 10% of the glutaminase activity associated with pig brain mitochondria was readily extractable by a variety of techniques but the remainder was very resistant to extraction. These two forms, which have been termed the soluble and membrane‐bound forms respectively, have been shown to differ in their responses to activation by phosphate and phosphate‐borate containing buffers. Submitochondrial fractionation studies indicated that the soluble form was located in the mitochondrial inner matrix whereas the membrane‐bound form was associated with the inner membrane. The mitochondria associated with the synaptosomes were found to contain only the membrane‐bound form of the enzyme whereas both forms were present in the free brain mitochondria.

Partial purification and characterization of a protein inhibitor of phosphoenolpyruvate carboxylase kinase

Abstract. The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoë fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L.was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.

Roles of Circadian Rhythms, Light and Temperature in the Regulation of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in the leaf tissue of CAM plants. It catalyses the nocturnal fixation of atmospheric CO2 (as HCO3 −) into oxaloacetate, which is subsequently reduced to malate and stored in the vacuole. During the day, malate released from the vacuole is decarboxylated and the resulting CO2 is fixed via the Calvin cycle (e.g. Osmond and Holtum 1981). Consideration of the metabolic pathways involved in CAM suggests that mechanisms must exist to permit flux through PEPC at night and reduce or eliminate it during the day. In this chapter we describe the role of phosphorylation in the regulation of PEPC in the CAM plant Bryophyllum (Kalanchoe) fedtschenkoi.