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.     

Induction of RNase and nuclease activity in cultured maize endosperm cells following sucrose starvation

The activity of RNases and nucleases in plants often increases following exposure to many types of stress, including prolonged exposure to dark or phosphate starvation. In cereals, the activity of RNases and nucleases is also regulated developmentally during late seed development. In this study, we investigated the effect that the absence of sugar or phosphate in culture medium has on the activity of RNases and nucleases expressed in maize endosperm suspension cells. Withdrawal of sugar from the culture medium resulted in a substantial increase in RNase and nuclease activities, whereas deprivation of phosphate during the same period of growth had no detectable effect on either of these activities. The increase in RNase activity was limited to the neutral RNases, demonstrating that the effect of sugar starvation is specific to one class of RNase. Elimination of asparagine from the medium resulted in a transient reduction in nuclease but not in RNase activity. These observations suggest that sugar starvation constitutes a stress to which maize endosperm responds, in part, by increasing neutral RNase and nuclease activity.     

Extracellular RNase produced by Yarrowia lipolytica

Production of extracellular RNase(s) by Yarrowia lipolytica CX161-1B was examined in media between pHs 5 and 7. RNase production occurred during the exponential growth phase. High-molecular-weight nitrogen compounds supported the highest levels of RNase production. Several RNases were detected in the supernatant medium. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the RNases had estimated molecular weights of 45,000, 43,000, and 34,000. It was found that Y. lipolytica secretes only one RNase (the 45,000-molecular-weight RNase) and that the 43,000 and 34,000-molecular-weight RNases are degradation products of this RNase. The alkaline extracellular protease secreted by Y. lipolytica was shown to have a major role in the 45,000- to 43,000-molecular-weight conversion, and it was demonstrated that the 45,000-molecular-weight RNase could be purified from a mutant which does not produce the alkaline extracellular protease. Purification of the RNase from a wild-type strain resulted in purification of the 43,000-molecular-weight RNase. This RNase was a glycoprotein with a molecular weight of 44,000 as estimated by gel filtration, an isoelectric point of pH 4.8, and a pH optimum between 6.5 and 7.0.     

The Effect of Water Stress on Floral Characters, Pollination and Seed Set in White Clover (Trifolium repens L.)

White clover plants were subjected to either short-term developingwater stress or long-term stable levels of water deficit. Thehort-term stress reduced plant water status to about –2·0MPa over 16 d. The long-term stress was less severe, but wassustained for several weeks. Long-term water stress promotedthe production of inflorescences. However, water stress alsoincreased floret abortion and the premature death of whole flowerheads. The number of ovules per floret was decreased by waterstress. The most striking effect of both long- and short-term waterdeficit was to reduce pollen viability measured with the fluorochromaticassay. This was not an artefact of assay conditions. The pollenfrom water-stressed flower heads was not reversibly dehydrated;it did not score at similar viability to controls after incubationin conditions which hydrate pollen. In addition, the pollenfrom water-stressed plants lost viability more rapidly thanpollen from well-watered plants after removal from the flowerhead. The consequences of reduced pollen viability on seed set wereinvestigated by hand-crossing within and between groups of plantsmaintained for several weeks at three levels of water supply.Flower heads pollinated with pollen from water-stressed plantsset fewer seeds per floret than those pollinated with controlpollen. Key words: Trifolium repens, white clover, water stress, floral characters, seed set     

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.     

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 and characterization of three ribonucleases from human kidney: comparison with urine ribonucleases

Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, amino-terminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to "nonsecretory" RNase, and that RNases HK-2A and HK-2B are both related to "secretory" RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.     

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.     

Drought-induced root aerenchyma formation restricts water uptake in rice seedlings supplied with nitrate

Previous studies demonstrated that ammonium nutrition results in higher water uptake rate than does nitrate nutrition under water stress, and thus enhances the tolerance of rice plants to water stress. However, the process by which water uptake is related to nitrogen form under water stress remains unknown. A hydroponic experiment with simulated water stress induced by polyethylene glycol (PEG6000) was conducted in a greenhouse to study the relationship between root aerenchyma formation and water uptake rate, such as xylem sap flow rate and hydraulic conductance, in two different rice cultivars (cv. 'Shanyou 63' hybrid indica and cv. 'Yangdao 6' indica, China). The results showed that root aerenchyma tissue increased in water-stressed plants of both cultivars fed by nitrate. No significant difference was found in root hydraulic conductivity and/or xylem sap flow rate between the two rice cultivars fed by ammonium regardless of water status, whereas these parameters decreased significantly in water-stressed plants fed by nitrate. It was concluded that aerenchyma that formed in the root cortex impeded the radial transport of water in the root cylinder and decreased water uptake in water-stressed rice plants fed by nitrate. Water transport occurred mainly through Hg-sensitive water channels in rice roots supplied with ammonium.     

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.

     

The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes.

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

EFFECTS OF WATER STRESS,ABSCISIC ACID,AND GIBBERELLIC ACID ON FLOWER PRODUCTION AND DIFFERENTIATION IN THE CLEISTOGAMOUS SPECIES COLLOMIA GRANDIFLORA DOUGL. EX LINDL. (POLEMONIACEAE)

The effects of water stress, abscisic acid (ABA), and gibberellic acid (GA₃) on flower production and differentiation by Collomia grandiflora were investigated. An untreated plant typically produced both small, closed cleistogamous (CL) and large, open chasmogamous (CH) flowers. The larger corolla of CH flowers was due to a greater cell number and size. When plants were water-stressed or sprayed with ABA, both the percentage of CH flowers and the total number of flowers were reduced significantly. The corolla dimensions and epidermal cell numbers and sizes of CL flowers produced by water-stressed and ABA-sprayed plants did not differ from those of CL flowers produced by control plants. Application of GA₃ to both well-watered and water-stressed plants significantly increased the percentage of CH flowers formed compared to well-watered controls. In the absence of GA₃, water-stressed plants produced almost entirely CL flowers. GA₃-sprayed plants produced CH flowers whose corolla dimensions were intermediate between those of CL and CH flowers formed by control plants. Epidermal cells of these intermediate corollas were reduced only in number and not in size when compared to control CH flowers. Endogenous levels of ABA and gibberellins may control the type of flower produced by C. grandiflora and may mediate some of the observable effects of water stress on flowering.     

Effects of Different Levels of Water Stress on Leaf Water Potential, Stomatal Resistance, Protein and Chlorophyll Content and Certain Anti-oxidative Enzymes in Tomato Plants

A greenhouse experlment was performed In order to Investigate the effects of dlfferent levels of water stress on leaf water potentlal （ψw）, stomatal resistance （rs）, protein content and chlorophyll （Chl） content of tomato plants （Lycoperslcon esculentum Mill. cv. Nlkita）. Water stress was Induced by addlng polyethylene glycol （PEG 6 000） to the nutrlent solution to reduce the osmotlc potential （ψs）. We Investlgated the behavlor of antl-oxldant enzymes, such as catalase （CAT） and superoxide dlsmutase （SOD）, durlng the development of water stress. Moderate and severe water stress （i.e. ψs= -0.51 and -1.22 MPa, respectlvely） caused a decrease In ψw for all treated （water-stressed） plants compared with control plants, wlth the reductlon belng more pronounced for severely stressed plants. In addltion, rs was slgnlflcantly affected by the Induced water stress and a decrease in leaf soluble protelns and Chl content was observed. Whereas CAT actlvlty remained constant, SOD actlvlty was increased in water-stressed plants compared wlth unstressed plants. These results Indicate the possible role of SOD as an anti-oxidant protector system for plants under water stress condltlons. Moreover, It suggests the possibllity of using this enzyme as an addltional screening crlterlon for detecting water stress in plants.     

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.     

Uniconazole-induced tolerance of soybean to water deficit stress in relation to changes in photosynthesis, hormones and antioxidant system

This study investigated whether uniconazole confers drought tolerance to soybean and if such tolerance is correlated with changes in photosynthesis, hormones and antioxidant system of leaves. Soybean plants were foliar treated with uniconazole at 50 mg L-1 at the beginning of bloom and then exposed to water deficit stress at pod initiation for 7 d. Uniconazole promoted biomass accumulation and seed yield under both water conditions. Plants treated with uniconazole showed higher leaf water potential only in water-stressed condition. Water stress decreased the chlorophyll content and photosynthetic rate, but those of uniconazole-treated plants were higher than the stressed control. Uniconazole increased the maximum quantum yield of photosystemand ribulose-1,5-bisphosphate carboxylase/oxygenase activity of water-stressed plants. Water stress decreased partitioning of assimilated 14C from labeled leaf to the other parts of the plant. In contrast, uniconazole enhanced translocation of assimilated 14C from labeled leaves to the other parts, except stems, regardless of water treatment. Uniconazole-treated plants contained less GA3, GA4 and ABA under well-watered condition than untreated plants, while the IAA and zeatin levels were increased substantially under both water conditions, and ABA concentration was also increased under water stressed condition. Under water-stressed conditions, uniconazole increased the content of proline and soluble sugars, and the activities of superoxide dismutase and peroxidase in soybean leaves but not the malondialdehyde content or electrical conductivity. These results suggest that uniconazole-induced tolerance to water deficit stress in soybean was related to the changes of photosynthesis, hormones and antioxidant system of leaves.     

Ribonuclease from the hepatopancreas of the red king crab <Emphasis Type="Italic">Paralithodes camtschatica</Emphasis>

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg²⁺ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT₅₀ = 53–55°C) and were strongly inhibited by NaCl (IC₅₀, 0.1–0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.     

Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> 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.