SummaryThe inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on speci®c metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose-6-phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT-and GPT-speci®c transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These ®ndings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3-phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM-induced), similar to non-green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic uxes during the diurnal CAM cycle.

In well-watered plants of Clusia uvitana, a species capable of carbon fixation by crassulacean acid metabolism (CAM), recently expanded leaves gained 5 to 13-fold more carbon during 12 h light than during 12 h dark periods. When water was withheld from the plants, daytime net CO uptake strongly decreased over a period of several days, whereas there was a marked increase in nocturnal carbon gain. Photosynthetic rates in the chloroplasts were hardly affected by the water stress treatment, as demonstrated by measurements of chlorophyll a fluorescence of intact leaves, indicating efficient decarboxylation of organic acids and refixation of carbon in the light. Within a few days after rewatering, plants reverted to the original gas exchange pattern with net CO uptake predominantly occurring during daytime. The reversible increase in dark CO fixation was paralleled by a reversible increase in the content of phosphoenolpyruvate (PEP) carboxylase protein. In wellwatered plants, short-term changes in the degree of dark CO fixation were induced by alterations in CO partial pressure during light periods: a decrease from 350 to 170 μbar CO caused nocturnal carbon gain, measured in normal air (350 μbar), to increase, whereas an increase to 700 μbar CO, during the day, caused net dark CO fixation to cease. The increased CAM activity in response to water shortage may, at least to some extent, be directly related to the reduced carbon gain during daytime.

Summary• The sink-source transition of developing Nicotiana tabacum (tobacco) leaves was studied here using chlorophyll fluorescence imaging.• In accordance with leaf development, the quantum efficiency of PSII, showed a steep gradient across the leaf with increasing values towards the tip.• The linear electron transport rate (ETR) saturated at higher CO 2 concentrations in the younger, than in the mature, part of the leaf, probably due to a lower Rubisco activity or a higher CO 2 diffusion resistance.• The induction of ETR at CO 2 concentrations near the compensation point after long-term dark adaptation of the young leaf, showed distinct responses; ETR rose rapidly in the basal but more slowly in the apical regions. There was a correlation between fast induction and carbohydrate import, as measured by 14 C-translocation. In the basal regions, larger pools of metabolic intermediates are expected due to imported carbohydrates. These might be used in the Calvin cycle directly after dark-light transition providing the electron acceptors for the faster induction of ETR. Additionally, a higher mitochondrial respiration can provide CO 2 for the Calvin cycle in these regions.

Phosphoenolpyruvate (PEP) carboxylation was measured as dark CO fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4-5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from darkadapted leaves of the common ice plant Mesembryanthemum crystallinum L . with crassulacean-acid metabolism (CAM). Purification by (NH&S04 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23 -25 U/mg protein and a protein kinase activity of255 pmol P,. mol-' PyrPC . s-'. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 pM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.-A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300 -3051 that Serll is the phosphorylation site of the CAM PyrPC of M . crystallinum. At pH 7.0, the K,,, for ATP of the protein kinase was 25 pM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone 111-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M . crystallinum, similar to C, and constitutive CAM plants.

Kreis M, Shewry PR, Halford NG (1991) Complementation of snfl, a mutation affecting global regulation of carbon metabolism in yeast, by a protein kinase cDNA. Proc Natl Acad Sci USA 88: Anderberg RJ, Walker-Simmons MK (1992) Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases. Proc Natl Acad Sci USA 8 9 10183-10187 Baur B, Dietz K-J, Winter K (1992) Regulatory protein phosphoryl-8602-8605 Baur et al. Plant Physiol. Vol. 106, 1994 ation of phosphoenolpyruvate carboxylase in the facultative CAM-plant Mesembyanthemum crystallinum L. Eur J Biochem Celenza JL, Carlson M (1986) A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science Cushman JC, Bohnert HJ (1994) Transcriptional activation of CAMgenes during development and environmental stress. h K Winter, AP Smith, JAC Smith, eds, Crassulacean Acid Metabolism, Biochemistry, Ecophysiology and Evolution. Springer-Verlag, Heidelberg, Germany (in press) Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52 Le Guen L, Thomas M, Bíanchi M, Halford NG, Kreis M (1992) Structure and expression of a gene from Arabidopis thaliana encoding a protein related to SNFl protein kinase. Gene 120 Ma H (1993) Protein phosphorylation in plants: enzymej, substrates 209 95-101 233 1175-1180 249-254 and regulators. Trends Genet 9 228-230 www.plantphysiol.org on July 15, 2020 -Published by Downloaded from