Gillian A. Nimmo

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 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.

Phosphorylation of phosphoenolpyruvate carboxylase plays a key role in the control of plant metabolism. Phosphoenolpyruvate carboxylase kinase is a Ca2+-independent enzyme that is activated by a process involving protein synthesis in response to a range of signals in different plant tissues. The component whose synthesis is required for activation has not previously been identified, nor has the kinase been characterised at a molecular level. We report the cloning of phosphoenolpyruvate carboxylase kinase from the Crassulacean Acid Metabolism plant Kalanchoë fedtschenkoi and the C3 plant Arabidopsis thaliana. Surprisingly, phosphoenolpyruvate carboxylase kinase is a member of the Ca2+/calmodulin-regulated group of protein kinases. However, it lacks the auto-inhibitory region and EF hands of plant Ca2+-dependent protein kinases, explaining its Ca2+-independence. Its sequence is novel in that it comprises only a protein kinase catalytic domain with no regulatory regions; it appears to be the smallest known protein kinase. In K. fedtschenkoi, the abundance of phosphoenolpyruvate carboxylase kinase transcripts increases during leaf development. The transcript level in mature leaves is very low during the photoperiod, reaches a peak in the middle of the dark period and correlates with kinase activity. It exhibits a circadian oscillation in constant conditions. Protein kinases are typically regulated by second messengers, phosphorylation or protein/protein interactions. Phosphoenolpyruvate carboxylase kinase is an exception to this general rule, being controlled only at the level of expression. In K. fedtschenkoi, its expression is controlled both developmentally and by a circadian oscillator.

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.

Inhibitor‐1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3′: 5′‐ monophosphate (cyclic AMP0, but not by phosphorylase kinase or by glycogen synthetase kinase‐2. Protein phosphatase‐III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor‐1. the kinetic constants for the dephosphorylation of inhibitor‐1 [Km= 0.7 μM, V (rel)=40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km = 1.1 μM, V (rel)= 62] and phosphorylase [Km = 5.0 μM, V (rel) = 100]. The dephosphorylation of inhibitor‐III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase‐III was diluted into buffers containing excess EDTA. it lost activity initially, but after 90 min, the activity reached a plateau that remainned stable for at least 20 h. The initial loss in activity varied with the substrate that was tested; it was 20‐30% with phosphorylase a, 50–60% with phosphorylase kinase and ≥ 95% with inhibitor‐1. This form of protein phosphatase‐III was inhibited by inhibitor‐1 in a noncompetitive manner, and the Ki for inhibitor‐1 was 1.6 ± 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase‐III were inhibited in an identical manner by inhibiter‐1. This result emphasizes the potential importance of inhibitor‐1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three defferent enzymes. The formation of the inactive complex between inhibitor‐1 and protein phosphatase‐III was reversed by incubation with trypsin (which destroyed inhibitor‐1, but not ptotein phosphatase‐III) or by dilution of the inactive complex. Kinetic studies, using the from of protein phosphatase‐III which dephosphorylated inhibitor‐1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor‐1 was still as powerful an inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor‐1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor‐1 even when it was present in a 50‐fold molar excess over inhibitor‐1. The result of these three properties is that inhibitor‐1 is preferentially dephosphorylated by prorein phosphatase‐III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor‐1 was also dephosphorylated by protein phosphatase‐II. The kinetic constants for the dephosphotylation of inhibitor‐1 [Km = 2.8 μM, V (rel) = 200] and the α‐subunit of phosphorylase kinase[Km = 3.7 μM, V (rel) = 100] were comparable. Inhibitor‐1 only inhibited protein phosphatase‐II by virtue of its ability to aact as an alternative substrate for the enzyme, and it inhibited protein phosphatase‐II at least several hundred‐fold less effectively than protein phosphatase‐III. The dephosphorylated form of inhibitor‐1 did not affect he activity jof protein phosphatase‐II or protein phosphatase‐III, nor did it affect the ability of protein phosphatase‐III to be inhibited by the phosphorylated form of inhibitor‐1, even when present in a 50‐fold molar excess over the phosphorylated form. Inhibitor‐1 was partially purified by a procedure in which the initial heat treatment at 90 °C was omitted. The behaviour of the protein on ion‐exchange chromatography, gel filtration and gel electrophoresis was identical to that of the protein prepared from heated extracts, and activation of the preparation by phosphorylation with cyclic‐AMP‐dependent protein kinase was accompanied by the forination of phosphothreonine. The results showed that inhibitor‐1 was not an artefact produced by heating muscle extracts at 90 °C. The evidence which indicates that the dephosphorylated form of inhibitor‐1 and protein phosphatase‐III do not form a complex, and the possibility that protein phosphatase‐III is the enzyme which dephosphorylates inhibitor‐1 in vivo are discussed.