Expression of Three RNase Activities during Natural and Dark-Induced Senescence of Wheat Leaves

We have monitored the activities of RNases WL_A, WL_B, and WL_C (A Blank, TA McKeon [1991] Plant Physiol 97: 1402-1408) during leaf senescence in wheat (Triticum aestivum L. cv Chinese Spring). When seedlings were induced to senesce in darkness, protein loss from primary leaves began immediately. RNase WL_B activity was unchanged for 2 days and then rose linearly, reaching a sixfold elevation in 7 days. RNase WL_C activity declined for 2 days and then rose linearly, reaching a twofold elevation in 7 days. RNase WL_A activity declined in the first 2 days and was unchanged thereafter. Although differentially expressed, these RNase activities may respond to a common regulatory mechanism(s) which, at 2 days of darkness, signals progression into a more advanced stage of senescence. The RNase activities were also differentially expressed during light-induced recovery, returning to normal levels in dissimilar patterns. In flag leaves of greenhouse-grown wheat, the three RNase activities increased during the early postanthesis period when protein content was stable and underwent further, accelerated accumulation during senescence. RNase WL_B activity showed the largest overall senescence-associated elevation (sixfold), followed by RNase WL_C (fourfold) and RNase WL_A (threefold).     

Purification and properties of two ribonuclease H enzymes from yeast

Two ribonuclease H activities have been purified from Saccharomyces cerevisiae. The major protein, RNase H_A is an acidic protein with a molecular weight of 65,000. RNase H_B is a basic protein with molecular weight of 54,000. Both RNases are active at alkaline pH range and require divalent cations for activity. RNase H_A has an absolute requirement for Mg²⁺, while Mn²⁺ can replace Mg²⁺ for RNase H_B. RNase H_A is inhibited by low concentrations of N-ethylmaleimide, whereas RNase H_B activity is unaffected under similar conditions. Substrate specificity studies using various polyribonucleotide · poly-deoxynucleotide hybrids showed that RNase H_A preferentially degrades polycytidylate, while RNase H_B is specific for polyadenylate. Kinetic analysis of the degradation of specifically end-labeled polymers and analysis of the products of the two yeast RNase H enzymes showed that yeast RNase H_A is an endonuclease producing 5′-phosphorylated oligonucleotides while yeast RNase H_B is a 5′-exonuclease producing 5′-AMP.     

Identification and Properties of the Major Ribonucleases of Arabidopsis thaliana

The profile of major ribonuclease (RNase) activities of Arabidopsis thaliana has been identified and characterized using a substrate-based gel assay. Following sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as many as 16 RNases, varying in size from 9 to 41 kilodaltons can be detected. Most of the RNase activities exhibit a pH optimum of about 6.5; however, the activity of a 22.6-kilodalton RNase is greatly enhanced at low pH. A number of the RNases in the 30- to 41-kilodalton range are sensitive to ethylenediaminetetraacetic acid, and their activities are enhanced by the presence of a low concentration of zinc during renaturation. At least one RNase appears to comigrate with a major DNase activity. The differential accumulation of several RNases in stems versus leaves indicates that some RNases are controlled in an organ-specific manner in A. thaliana.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Distribution of a Kidney Acid-ribonuclease-like Enzyme and the Other Ribonucleases in Bovine Organs and Body Fluids

To determine the distribution of a kidney acid RNase (RNase K₂) and other RNases, the levels of RNase K₂, RNase A, and seminal RNase (RNase Vs₁ in bovine tissues and body fluids were measured by enzyme immunoassay. The crude extracts of several tissues and body fluids were fractionated by phospho-cellulose column chromatography. The enzymatic activities at pH 7.5 and 6.0 and enzyme contents of each tube were measured by enzyme assay and enzyme immunoassay, respectively. In the pancreas, parotid gland, and heart, most RNase activity was due to a single peak of RNase A, but a small amount of RNase K₂ was always observed. In the kidney, there was about 5 times as much RNase K₂ as RNase A. In the lung, although RNase K₂ and RNase A were the major components, there are another two alkaline RNase peaks. In the spleen and liver, there are four RNases, two acid RNases, one of which is RNase K₂, and two alkaline RNases including RNase A. A new acid RNase (non RNase K₂-acid RNase) from both organs was immunologically the same. In serum, there are at least four RNases. By partial purification of serum RNases by phosphocellulose and heparin-Sepharose column chromatographies, at least 4 RNases, RNase A, RNase K₂ and the other two alkaline RNases, one of which is immunologically indistinguishable from liver alkaline RNase, were confirmed. The other serum alkaline RNase was immunologically related to lung and spleen alkaline RNases. In conclusion, in bovine tissues and body fluids there are at least 7 types of pyrimidine-base-specific RNases: brain RNase, seminal RNase, RNase A, RNase K₂, an acid RNase (RNase BSPJ, an alkaline RNase (RNase BL₄), and another alkaline RNase in serum.     

Purification and properties of two ribonucleases in different intracellular compartments in pea root tissue.

Two RNases in bound forms associated with the microsomal membrane and with the ribosomes or unknown particles in pea root tissue were solubilized by subjecting the membrane to sonic oscillation in the presence of EDTA and KC1 and by treating the particles with EDTA, respectively. The RNases were than purified by DEAE-cellulose and Sephadex G-75 column chromatographies. The elution profiles of RNases from the columns were very similar. No significant differences were observed in their electrophoretic mobilities in polyacrylamide gels, in molecular weight, in activation by inorganic ions, urea or phospholipid micelles or in the dependence of their activities upon pH. The purified RNASES were not different from the bound enzymes as regards activation by inorganic ions and urea and the dependence of the activity upon pH. Triton X-100 stimulated the activity only if RNase was in a bound form associated with the microsomal membrane. We propose that the two RNases may be the same molecular species and differ only in the form of association with intracellular structures.     

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.     

Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2′, 5′-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR_A and GR_B). Purified GR_A had a specific activity of 1.81 microkatals per milligram of protein and GR_B had a specific activity of 6.08 microkatals per milligram of protein. GR_A accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR_B accounted for 83%. The native molecular mass for GR_A was 103 to 104 kilodaltons and for GR_B was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR_A and 57 kilodaltons for GR_B. The pH optimum for GR_A was 7.25 to 7.75 and for GR_B was 7.25. At 25°C the K_m for GSSG was 15.3 and 39.8 micromolar for GR_A and GR_B, respectively. For NADPH, the K_m was 3.7 and 8.8 micromolar for GR_A and GR_B, respectively. Antibody produced from purified GR_B was reactive with both native and denatured GR_B, but was cross-reactive with only native GR_A.     

Identification of a plant-specific Zn<Superscript>2+</Superscript>-sensitive ribonuclease activity

Ribonucleases (RNases) play a variety of cellular and biological roles in all three domains of life. In an attempt to perform RNA immuno-precipitation assays of Arabidopsis proteins, we found an EDTA-dependent RNase activity from Arabidopsis suspension tissue cultures. Further investigations proved that the EDTA-dependent RNase activity was plant specific. Characterization of the RNase activity indicated that it was insensitive to low pH and high concentration of NaCl. In the process of isolating the activity with cation exchange chromatography, we found that the EDTA dependency of the activity was lost. This led us to speculate that some metal ions, which inhibited the RNase activity, may be removed during cation exchange chromatography so that the nuclease activity was released. The EDTA dependency of the activity could be due to the ability of the EDTA chelating those metal ions, mimicking the effect of the cation exchange chromatography. Indeed, Zn²⁺ strongly inhibited the activity, and the inhibition could be released by EDTA based on both in-solution and in-gel assays. In-gel assays identified two RNase activity bands. Mass spectrometry assays of those activity bands revealed more than 20 proteins. However, none of them has an apparent known nuclease domain, suggesting that one or more of those proteins might possess a currently uncharacterized nuclease domain. Our results may shed light on RNA metabolism in plants by introducing a novel plant-specific RNase activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specific RNase isoenzymes in the human central nervous system

After inactivation of RNase inhibitor by parachloromercuribenzoate, total alkaline RNase activity was found to be two fold higher in white matter as in grey matter extracts from human brain tissue. This activity was lower in human purified myelin. Two human cerebrospinal fluid (CSF) RNase isoenzymes of group 3 (a minor one, RNase 3.1, and a major one, RNase 3.2) were found to be present in human grey and white matter extracts and in purified myelin, but absent in human serum, peripheral nerve, liver, and spleen extracts. A RNase isoenzyme similar to central nervous system (CNS) RNase 3.2 was present in human kidney extracts but it differed in its carbohydrate structure. RNase isoenzymes 3.1 and 3.2 were not found in mouse, rat, and bovine brains. Thus, RNases 3.1 and 3.2 seem specific to human CNS. RNases of group 3 are the predominant RNase isoenzymes in CSF and one of the two predominant RNase groups in brain tissue. However, the proportion of RNases of group 3 is different in CSF and in brain extracts: RNases 3.1-3.2 are the major constituents of group 3 RNases in brain tissue, while another RNase isoenzyme of group 3, RNase 3.0, which is more glycosylated than RNases 3.1-3.2, is only a minor part of RNase of group 3 in brain extracts. Conversely, RNases 3.1-3.2 are lower or equivalent to RNase 3.0 in control CSF since the ratio of RNases 3.1-3.2 to RNase 3.0 did not exceed 1.0. This ratio decreased in pathological CSF including multiple sclerosis or infectious CNS diseases that were free of transudation phenomena. In conclusion, CSF RNases 3.1-3.2 seem to originate in brain tissue and could be markers of RNA catabolism from brain cells.     

Expression of Nicotiana glutinosa Ribonucleases in Escherichia coli

We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively. In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity. RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I). Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU. Furthermore, kinetic parameters for RNase NW (K _m, 0.778 mM and k _cat, 1938 min^?1) and RNase NGR3 (K _m, 0.548 mM and k _cat, 408 min^?1) were calculated using GpU as a substrate.     

RNase Activity Decreases following a Heat Shock in Wheat Leaves and Correlates with Its Posttranslational Modification

Heat shock results in a coordinate loss of translational efficiency and an increase in mRNA stability in plants. The thermally mediated increase in mRNA half-life could be a result of decreased expression and/or regulation of intracellular RNase enzyme activity. We have examined the fate of both acidic and neutral RNases in wheat seedlings that were subjected to a thermal stress. We observed that the activity of all detectable RNases decreased following a heat shock, which was a function of both the temperature and length of the heat shock. In contrast, no reduction in nuclease activity was observed following any heat-shock treatment. Antibodies raised against one of the major RNases was used in western analysis to demonstrate that the RNase protein level did not decrease following a heat shock, and the data suggest that the observed decrease in RNase activity in heat-shocked leaves may be due to modification of the protein. Two-dimensional gel/western analysis of this RNase revealed three isoforms. The most acidic isoform predominated in control leaves, whereas the most basic isoform predominated in leaves following a heat shock and correlated with the heat-shock-induced reduction in RNase activity and increase in mRNA half-life. These data suggest that RNase activity may be regulated posttranslationally following heat shock as a means to reduce RNA turnover until recovery ensues.     

Free Fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves

Effects of MgCl₂ and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl₂ with a saturated concentration of more than 5 millimolar. Free α-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl₂, without affecting its maximum activity at higher MgCl₂ concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl₂ were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl₂, CaCl₂ and a high concentration of KCl in place of MgCl₂. UDGT activity was hyperbolically enhanced by MgCl₂ with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl₂-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl₂ concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.     

Influence of ozone on ribonuclease activity in wheat (Triticum aestivum) leaves

Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (P(i)) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O(3)) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O(3). Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O(3) mol(-1)) (control) or non-filtered air plus O(3) (90 nmol O(3) mol(-1)) (O(3)) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O(3)-sensitive cultivar in the O(3) treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [P(i)]. Soluble [P(i)] in leaf tissue extracts from the O(3) and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O(3) treatment. Isozymes stimulated in the O(3) treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [P(i)] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O(3) were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [P(i)] in the leaf tissue samples suggested that O(3)-induced injury and accelerated senescence was atypical of normal senescence processes in that P(i) export was not observed in O(3)-treated plants.     

Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

Changes in Ribonuclease Activity of Wheat Plants during Water Stress

RNases from control and water-stressed wheat seedlings were purified by ion exchange chromatography, gel filtration and gel electrophoresis. The enzyme appeared to be identical from both treatments and only one species was found. Subcellular distribution between soluble and particulate fractions was not changed by water stress treatment. Total quantity of RNase per plant was similar in control and water-stressed plants, although since the dry weight of treated plants was less, the activity per gram was significantly higher. Crude homogenates of leaf extracts contained an RNase inhibitor with a greater amount being present in control leaves. No evidence was found to indicate that new types of RNases are synthesized during water stress.     

Pyrroline-5-Carboxylate Reductase Is in Pea (Pisum sativum L.) Leaf Chloroplasts

Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Δ¹-Pyrroline-5-carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl₂. The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8.     

Purification and Properties of a Ribonuclease from Cowpea Cotyledons

The isolation and characterisation of cotyledonary ribonucleases (RNase; EC 3.1.27.1), are basic steps to understand the physiology and biochemistry of RNA turnover and mobilisation during seed germination and seedling establishment, as well as how environmental stresses affect them. RNase was isolated and purified 928-fold, to apparent electrophoretic homogeneity from 5-d-old seedlings of Vigna unguiculata. It is a protein with an apparent molecular mass of 16 kDa having three major isoforms. Its optimum pH is 5.8, which decreases to 5.2 in presence of KCl. It has an apparent K_m of 0.80 mg RNA cm^-3 and retains 40 % of its activity when heated to 80 °C. It is completely inhibited by Cu²⁺, Hg²⁺ and Zn²⁺ and is almost insensitive to Mg²⁺, Ca^2+- and EDTA. Urea, Fe²⁺, Co²⁺ and 2-mercaptoethanol partially inhibit its activity. Its amino acid composition shows a resem lance to that of other plant RNases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.