Andre T. Jagendorf

Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice

Abstract Higher plants growing in natural environments experience various abiotic stresses. H2O2 and nitric oxide (NO) free radicals are produced and cause oxidative damage to plants under various abiotic stress conditions. However, in the present study, we found that pretreating rice seedlings with low levels (

Artificial reductant enhancement of the Lowry method for protein determination

Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used.

RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance

Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the cold-regulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance.

Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance

The Arabidopsis Salt Overly Sensitive 2(SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensorSalt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168DΔF, and SOS2T168DΔ308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn2+ relative to Mg2+ and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168DΔ308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.

ATP-dependent proteolysis in pea chloroplasts

Proteins newly formed from labeled amino acids by isolated intact pea chloroplasts are not entirely stable. Between 20 and 35% of the labeled protein is degraded over a 20–30 min incubation period in pulse‐chase experiments. Protein degration is prevented when chloroplast ATP level drops, as in the dark without added ATP. Degration is stimulated by adding ATP directly or by generating it in photophosphorylation. Susceptible new proteins are not stabilized against further additions of ATP, during incubation under ATP‐deficient conditions.

A Purified Zinc Protease of Pea Chloroplasts, EP1, Degrades the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

A previously reported endopeptidase (EP1) from pea chloroplasts was purified over 11,000-fold using a four-step protocol involving ultrafiltration, sucrose gradient centrifugation, isoelectric focusing, and high performance liquid chromatography gel filtration. The enzyme was determined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chloroplasts was confirmed by demonstrating its insensitivity to thermolysin when the envelope was intact. A contaminating serine protease that attacks EP1 was found. The contaminating protease was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, whereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygenase under physiological conditions.

Effect of methanol on spinach thylakoid ATPase

Abstract Methanol at 35% ( v v ) overcomes the latency of spinach thylakoid ATPase. Activation is immediate and reversible involving changes in the V max , not the K m of the enzyme, MgATP is a much better substrate than CaATP; free Mg 2+ noncompetitively inhibits activity. This inhibition can be overcome by the addition of Na 2 SO 3 . While both MgATP and MgGTP act as substrates, free ATP and GTP both inhibit activity. ADP and MgADP are also inhibitory. Insensitivity to certain inhibitors indicates that methanol neither induces the same conformational changes in CF 1 as illumination does, nor does it lead to coupling between H + movement through CF 0 and ATP hydrolysis. Methanol activation provides a much improved method for assaying thylakoid ATPase.

Ionic and pH transitions triggering chloroplast post-illumination luminescence☆

Abstract The postillumination “chemiluminescence” of isolated spinach chloroplasts, previously shown to be caused by an acid-base transition, is found to be caused also by a transition from a low to a high ionic strength environment. The salt induction of luminescence is in competition with the acid-base luminescence; whichever comes first prevents light emission upon going through the second process. It was discovered that suddenly lowering the pH to 3.2 or below also causes light emission. The acid procedure induces up to eight times as much light emission as the others, and does not seem to compete with them in that raising the pH or the salt concentration causes a further amount of luminescence even after the acid-induced burst. All three procedures share a sensitivity to phosphorylation inhibitors and to DCMU, and all require preillumination of the chloroplasts. Chloroplasts subject to a low to high salt transition do not go into a high energy state, as judged by either ATP formation or ATPase activation. Evidence is presented suggesting that even in the case of acid-base transition the high energy state is not the trigger for light emission.

Immunological identification of nascent subunits of wheat ribulose diphosphate carboxylase on ribosomes of both chloroplast and cytoplasmic origin.

Abstract Wheat ribulose diphosphate carboxylase was dissociated into subunits by denaturation in the presence of guanidine hydrochloride and p -chloromercuribenuoate followed by separation of the subunits on Sephadex G-75 in the presence of urea. After removal of the mercurial reagent, the subunits were injected into rabbits, and monospecific antisera to the two different subunit types were obtained. High-titer antiserum to the native ribulose diphosphate carboxylase was also obtained. Specific antibody precipitation of labeled polypeptides, formed by 3 H]puromycin incubation with wheat ribosomes, was used to localize ribulose diphosphate carboxylase nascent chains on the chloroplast and cytoplasmic ribosomes. Only soluble peptidyl-[ 3 H]puromycins actually released from the ribosomes were assayed: bound peptidyl-[ 3 H]puromycins were either removed by sedimentation or solubilized by neutral detergent prior to analysis, with identical results in each case. Assays were performed on individual sucrose gradient fractions or on highly purified 70S and 80S monoribosome preparations with comparable results: with ribosomes from 4-day seedlings, for instance, 30% of peptidyl-[ 3 H]puromycins made by 70S ribosomes reacted with antibody to the native enzyme or its large subunit, while 5% of peptidyl-[ 3 H]puromycins made by 80S ribosomes reacted with antibody to the large subunit, the small subunit, or the native enzyme, in a nonadditive manner. The results suggest that chloroplast ribosomes make the large, but not the small subunit of the enzyme, while 80S ribosomes make the small subunit, which associates with completed large subunits while still on the ribosomes.

Treatment of Pea (Pisum sativum L.) Protoplasts with DNA-Damaging Agents Induces a 39-Kilodalton Chloroplast Protein Immunologically Related to Escherichia coli RecA

Organisms must have efficient mechanisms of DNA repair and recombination to prevent alterations in their genetic information due to DNA damage. There is evidence for DNA repair and recombination in plastids of higher plants, although very little is known at the biochemical level. Many chloroplast proteins are of eubacterial ancestry, suggesting that the same could be true for the components of a DNA repair and recombination system. A 39-kD protein, immunologically related to Escherichia coli RecA, is present in chloroplasts of pea (Pisum sativum L.). Bandshift gel assays suggest that it binds single-stranded DNA. Its steady-state level is increased by several DNA-damaging agents. These results are consistent with it being a plastid homolog of E. coli RecA protein, presumably involved in DNA repair and recombination, and with the existence of an SOS-like response in pea leaf cells. Experiments with protein synthesis inhibitors suggest that the 39-kD chloroplast protein is encoded in the nucleus.