Search

Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 µM) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg2+ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg2+. Subsequent addition of either Mg2+, Mn2+ or Ca2+ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg2+ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg2+-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg2+ -and Ca2+-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca2+ /Mg2+-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg2+ or Ca2+). It is tentatively suggested that Mg2+ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg2+ had no effect. However, 5'-AMP was a potent activator of the reaction with an apparent Km of 0.5 mM. There was also considerable specificity for 5'AMP relative to 3'- or 2'-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5'-AMP) or physical (e.g. cytosolic [Mg2+]) in nature.

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.

When spinach leaf tissue was subjected to evaporative dehydration, photosynthetic capacity at very high (5%) CO2 concentration and saturating irradiance (300 W·m-2), decreased in parallel to the relative water content (RWC). A 50% inhibition was observed at 60–40% RWC. In order to examine whether the inhibition was caused by increased solute concentrations in chloroplasts or cytoplasm, an artificial stroma medium (ASM) was set up containing all major osmotically relevant solutes measured in isolated intact spinach chloroplasts. Subsequently, the response of enzyme activities to normal and to increased concentrations of ASM was examined. Inhibition of enzymes by a concerted increase of all solutes was well correlated to the in-vivo response of photosynthesis to dehydration (60% inhibition at double-strength ASM). Inhibitory solutes were mainly divalent inorganic anions, such as sulfate and phosphate. Inhibition of ribulose-1,5-bisphosphate carboxylase by these ions as studied in more detail. Inhibition of the enzyme by sulfate and phosphate was competitive with respect to ribulose-1,5-bisphosphate, but not with respect to CO2. The KI for sulfate was 2.1 mmol·l-1 and for phosphate 0.57 mmol·l-1. Sugars and amino acids at the concentrations found in spinach chloroplasts did not prevent inhibition of enzymes by anions. The results indicate that increased anion concentrations in cells and organelles are responsible for primary, quickly reversible effects of moderate dehydration on plant tissues.

The conversion of U-labelled [14C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.

The effect of osmotic dehydration on metabolic reactions in three different subcellular compartments (chloroplast, cytoplasm and mitochondria) was studied in vacuum-infiltrated thin leaf slices from various plants, in the absence of stomatal control. The reactions tested were CO2 fixation in the light (chloroplast), CO2 fixation in the dark (cytoplasm), and O2 uptake in the dark (mitochondria). In most plants, the sensitivity of dark CO2 fixation to dehydration was similar to the sensitivity of photosynthesis. In leaf slices from a plant with Crassulacean acid metabolism (Kalanchoe pinnata), dark CO2 fixation (which reached similar rates as light fixation) was slightly more sensitive to osmotic stress than photosynthesis. Dark respiration (measured as O2 uptake) was significantly more resistant to hypertonic stress than both types of CO2 fixation. In crude leaf extracts from spinach, the response of soluble enzymes from the three different subcellular compartments to high concentrations of various electrolytes and neutral compounds was examined and compared with the in-vivo data.