While exposure of C₃ plants to elevated CO₂ would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the ...present study suggest that exposure of plants to elevated CO₂ can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated CO₂. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated CO₂. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated CO₂ or O₃ revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated CO₂.
Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in ...vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.
The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 ...weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance.
Sucrose is formed in the cytoplasm of leaf cells from triose phosphates exported from the chloroplast. Flux control is shared among key enzymes of the pathway, one of which is sucrose-phosphate ...synthase (SPS). Regulation of SPS by protein phosphorylation is important in vivo and may explain diurnal changes in SPS activity and carbon partitioning. The signal transduction pathway mediating the light activation of SPS in vivo appears to involve metabolites and novel "coarse" control of the protein phosphatase that dephosphorylates and activates SPS. Regulation of the phosphorylation of SPS may provide a general mechanism whereby sucrose formation is coordinated with the rate of photosynthesis and the rate of nitrate assimilation. There are apparent differences among species in the properties of SPS that may reflect different strategies for the control of carbon partitioning. The SPS gene has recently been cloned from maize; results of preliminary studies with transgenic tomato plants expressing high levels of maize SPS support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose.
Experiments were conducted to determine whether sucrose synthase (SuSy) was phosphorylated in the elongation zone of maize (Zea mays L.) leaves. The approximately 90-kD subunit of SuSy was ...32P-labeled on seryl residue(s) when excised shoots were fed 32Porthophosphate. Both isoforms of SuSy (the SS1 and SS2 proteins) were phosphorylated in vivo, and tryptic peptide-mapping analysis suggested a single, similar phosphorylation site in both proteins. A combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and automated Edman sequencing analysis unequivocally identified the phosphorylation site in the maize 552 protein as serine-15. This site was phosphorylated in vitro by endogenous protein kinase(s) in a strictly Ca2+-dependent manner. A synthetic peptide, based on the phosphorylation site sequence, was used to identify and partially purify an endogenous Ca2+-dependent protein kinase(s) from the maize leaf elongation zone and expanding spinach leaves. Phosphorylation of SuSy in vitro selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme for sucrose and UDP, suggesting that phosphorylation may be of regulatory significance. Conservation of the phosphorylation site, and the sequences surrounding it, among plant species suggests that phosphorylation of SuSy may be widespread, if not universal, in plants
The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and ...threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.
The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of ...evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant
Arabidopsis GF14ω, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14ω.
In this report we address two questions regarding the regulation of phosphorylated nitrate reductase (pNR; EC 1.6.6.1) by 14-3-3 proteins. The first concerns the requirement for millimolar ...concentrations of a divalent cation in order to form the inactive pNR:14-3-3 complex at pH 7.5. The second concerns the reduced requirement for divalent cations at pH 6.5. In answering these questions we highlight a possible general mechanism involved in the regulation of 14-3-3 binding to target proteins. We show that divalent cations (e.g. Ca(2+), Mg(2+) and Mn(2+)) bind directly to 14-3-3s, and as a result cause a conformational change, manifested as an increase in surface hydrophobicity. A similar change is also obtained by decreasing the pH from pH 7.5 to pH 6.5, in the absence of divalent cations, and we propose that protonation of amino acid residues brings about a similar effect to metal ion binding. A possible regulatory mechanism, where the 14-3-3 protein has to be "primed" prior to bi
Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton ...fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.
Postemergence (POST) application of 4‐hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors in combination with a photosystem II (PSII) inhibitor, such as atrazine ...6‐chloro‐N‐ethyl‐N9‐(1‐methylethyl)‐1,3,5‐triazine‐2,4‐diamine, is common practice in sweet corn (Zea mays L.) production. Given the sensitivity of sweet corn to HPPD‐inhibiting herbicides, the objective of this work was to determine the extent to which cytochrome P450 (CYP) genotype and PSII‐inhibitors affect crop sensitivity to HPPD‐inhibiting herbicides. Greenhouse experiments were used to identify PSII‐inhibitors that were least injurious when combined with the HPPD‐inhibitors, mesotrione 2‐(4‐mesyl‐2‐nitrobenzoyl)‐3‐hydroxycylohex‐2‐enone, tembotrione {2‐2‐chloro‐4‐(methylsulfonyl)‐3‐(2,2,2‐trifluoroethoxy)methylbenzoyl‐1,3‐cyclohexanedione}, and topramezone {3‐(4,5‐dihydro‐3‐isoxazolyl)‐2‐methyl‐4‐(methylsulfonyl)phenyl(5‐hydroxy‐1‐methyl‐1H‐pyrazol‐4‐yl)methanone}. Subsequently, HPPD‐inhibitors were tested individually with PSII‐inhibitors atrazine, or bentazon 3‐(1‐methylethyl)‐(1H)‐2,1,3‐benzothiadiazin‐4(3H)‐one2,2‐dioxide, or alone in field experiments on all three CYP genotypic classes; hybrids homozygous for mutant CYP alleles (cypcyp), hybrids homozygous for functional alleles (CYPCYP), and heterozygous hybrids (CYPcyp). Leaf bleaching within 1 wk of herbicide application increased when a PSII‐inhibitor was combined with an HPPD‐inhibitor; however, the relatively low level of injury was short‐lived. Tank mixing atrazine to mesotrione, tembotrione, or topramezone in sweet corn did not increase risk of yield loss compared to HPPD‐inhibitor applied alone. The synergistic effect on weed control between certain PSII‐ and HPPD‐inhibitor combinations reported previously does not hold true regarding sweet corn sensitivity to these herbicides. Among three HPPD‐inhibitors tested in sweet corn, topramezone was the safest, regardless of PSII combination. Mutant CYP alleles, namely CYPcyp and cypcyp hybrids, are the main cause of sweet corn sensitivity to mesotrione, tembotrione, and other CYP‐metabolized herbicides; therefore, breeding efforts to eliminate mutant CYP alleles should remain a high priority.
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