Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

RNase in Lasallia hispanica and Cornicularia normoerica: multiplicity of electromorphs and activity changes during a hydration-dehydration cycle

The presence of RNase activity has been detected in the twosaxicolous lichen species, Lasallia hispanica (Frey) Sancho& Crespo and Cornicularia normoerica (Gunn.) DR. Activitywas localized in the soluble fraction and had an acid optimumpH in both species. When proteins from the soluble fractionof the two lichens were separated by isoelectric focusing, multipleelectromorphs with RNase activity were detected. L. hispanicaRNase was separated into seven bands, characterized by pls 7,6.28, 4.58, 4.45, 4.25, 3.95, and 3.47. In C. normoerica fourbands were detected, with pls of 6.28, 3.98, 3.57, and 3.39.The molecular mass of the main RNase of L. hispanica estimatedby SDS-PAGE was 31.86 kDa, which corresponds to the 33 kDa estimatedfor the undenatured RNase by gel chromatography. Proteins fromC. normoerica were resolved by SDS-PAGE in three bands, withestimated molecular mass of 36.07 kDa, 31.86 kDa and 17.13 kDa.In order to improve the detection of RNase activity, gels wereincubated after the run (electrophoresis or isoelectric focusing)in a RNA solution, instead of including the substrate in thegel. In both species, RNase activity increased during hydrationand decreased during desiccation. This pattern of activity resemblesthat of other enzyme activities in lichens, which decrease inresponse to water deficits, and is different from the responseof other poikilohydrous organisms such as bryophytes. Theseresults are discussed in relation to the mechanisms that lichenshave to withstand dehydration. Key words: Comicularia, Lasallia, lichens, ribonuclease, water stress     

Human non-secretory ribonucleases. II. Structural characterization of theN-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry

The N-glylycans have been removed by peptide-N-glycosidase F(PNGase F) from purified human non-secretory RNases derivedfrom kidney, liver and spleen. The spleen RNase was purifiedby two procedures, one of which did not include the usual acidtreatment step (0.25 M H₂SO₄, 45 min, 4C), to determine ifacid treatment alters the carbohydrate moieties. TheN-glycansof the RNases were fractionated by Bio-Gel P-4 chromatographyand analysed by 600 MHz ¹H-NMR spectroscopy and electrospraymass spectrometry. All four non-secretory RNase preparationscontained the following structures: The relative amounts of the trisaccharide, pentasaccharide andhexasaccharide appeared to vary slightly in the different tissueRNases. The overall results indicate: (i) that acid treatmentduring purification does not alter the N-glycans of non-secretoryRNases; (ii) that the N-glycans from kidney, liver and spleennon-secretory RNases are very similar, if not identical, toone another, but different from the N-glycan structures reportedfor secretory RNase. N-glycans non-secretory RNases     

Morphology, RNase and transaminase of root protoplasts

RNase and transaminase activities were analysed for mechanicallyand enzymatically prepared protoplasts from Allium Cepa roots.The comparative analyses at three root regions show that theenzymes were less active in the protoplasts than in the cellsfrom which they had been obtained. The enzyme gradients (fromapex to base of the root: RNase increase and transaminase decrease)noted previously in the intact roots were found to be similarin the protoplasts, however to a lesser degree. On the otherhand, the relative activity of both tested enzymes was lowerin the enzymatically prepared protoplasts than in those obtainedby the mechanical technique. In connection with their physiologicalproperties, the respective effects of the mode of preparingthe protoplasts were discussed. (Received November 22, 1971; )     

Deuteration Causes the Decreased Induction of Heat-Shock Proteins and Increased Sensitivity to Heat Denaturation of Proteins in Chlorella

Deuterated Chlorella (D-Chlorella) cells were obtained by cultivationin a medium that contained D₂O. The modification of cell componentsby deuterium (D) increased the heat sensitivity of cells, whichdepended on the extent of deuteration. To elucidate the mechanismof the D-induced increase in the heat sensitivity, the effectof incorporation of D on the denaturation of proteins was investigated.Proteins obtained from D-Chlorella cells, when preheated at37°C, were aggregated to a greater extent than those fromH-Chlorella against the heating at 45°C. The rate of synthesisof heat-shock proteins (hsps) in D-Chlorella was consistentlylower than that in Hcells. Furthermore, an experiment in vitroindicated that deuterated proteins denatured more rapidly thannormal proteins upon heating at 60°C. (Received August 12, 1993; Accepted November 30, 1993)     

Ribonuclease and Other Factors Involved in the Respiratory Senescence of Maize Scutellum

The scutella from seedlings of Zea mays L. germinated at 28–30°C increase in respiration rate to the third day, followed bya decline which is quite noticeable by the fifth day. A searchhas been made for factors responsible for the respiratory decline.The electronmicroscope shows the five-day mitochondria to benormal in appearance. Very active preparations are obtainedby isolating the mitochondria at pH 7.6 with inclusions of bovineserum albumin and ethylenediamine-tetraacetic acid. A solubleribonuclease (RNase A) which increases rapidly with age impairsboth oxidation and phosphorylation. The largely particle-boundribonuclease (RNase B) is not inhibitory. Plant ribonucleaseis resistant to the proteolysis occurring during senescence.It is suggested that the soluble ribonuclease contributes tothe respiratory decline, but that other factors may also beinvolved.     

Ribonuclease activity of sialic acid-binding lectin from Rana catesbeiana eggs

Sialic acid-binding lectin (SBL) isolated from Rana catesbeianaeggs is a basic protein which agglutinates a large variety oftumour cells and has an amino acid sequence homologous to thatof human angiogenin and pancreatic ribonuclease (RNase). AlthoughSBL and angiogenin lack the Cys-65-Cys-72 disulphide bond ofpancreatic RNase, the locations of the other three disulphidebonds are similar among the three molecules. SBL was found toexhibit RNase activity, as well as catalytic properties resemblingthose of bovine RNase A in some respects. For example, SBL hydrolysespoly(uridylic acid) and poly(cytidylic acid) as substrates,and prefers the former. RNase A and angiogenin are stronglyinhibited by human placental RNase inhibitor, whereas the RNaseactivity and tumour cell agglutination activity of SBL are notaffected by this inhibitor.     

Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.     

The Development of Mitochondria in Arum Spadix

Electron micrographs of Arum spadix cells prepared at four stagesof development all showed abundant sections of mitochondriawith tubular ingrowths (microvilli). At the earliest stage (whenthe spadix cells were still dividing) there was an average of9 sections of microvilli per mitochondrion. In later stagesof development, when the cells were growing by elongation, thenumber of microvilli rose to 22. It was reported earlier that the succinoxidase activity of themitochondria of Arum spadix increased as the spadix developed,and it is now shown that the rise in enzyme activity parallelsthe increase in length of microvilli.     

Early Light-Response of psaD, psaE and psaH Gene Families of Photosystem I in Nicotiana sylvestris: PSI-D has an Isoform of Very Quick Response

Light response of the psaD, psaE and psaH gene families of photosystemI was studied in etiolated seedlings and dark-adapted matureleaves of Nicotiana sylvestris using RNase protection assaysand immunoblot analysis. The results suggest that the initiallight-response consists of at least two types; a very quickresponse found only in the psaDb gene, and a slower responsecommon to all the examined genes. (Received November 14, 1994; Accepted March 1, 1995)     

A Comparison of the Heat Pulse Method and Deuterium Tracing Method for Measuring Transpiration from Eucalyptus grandis Trees

Estimates of transpiration obtained with the heat pulse velocity(HPV) method and the deuterium tracing method were comparedin two Eucalyptus grandis Hill ex Maiden trees aged 18 monthsand 4 years, respectively. The HPV estimate of weighted meandaily flow rate differed from the deuterium mean by 7% in theolder tree, and 35% in the younger tree. An analysis of possiblesources of error revealed that the discrepancy in the youngertree may have arisen as a result of inadequate probe espacementradially through the sapwood, or from sapwood disruption causedwhen holes were drilled to release the tracer. We conclude thatthe two methods can provide very similar estimates of mean weightedtranspiration rate in E. grandis trees if due precautions aretaken to minimize known sources of error. The relative meritsof the two techniques, which in many respects are complementary,are discussed. Key words: Eucalyptus grandis, heat pulse velocity, deuterium, tracer, transpiration     

Messenger and ribosomal RNA hydrolysis by ribonucleases II. Changes in ribonuclease activities and ribosomes of barley leaves during the early stages of powdery mildew infection

We have monitored changes in the properties of two barley (Hordeumvulgare L.) leaf RNases with respect to their action on polysomalmessenger RNA (mRNA) and the RNA of isolated ribosomes duringthe early stages of infection by the powdery mildew fungus (Erysiphegraminis f. sp. hordei). The results presented support the followingconclusions. (i) At 48 hr after inoculation, the pH 5 insolubleRNase undergoes significant changes in its catalytic properties.This is evident from the finding that under limit digestionconditions, the enzyme from inoculated leaves hydrolyzes chloroplastpolysomal mRNA and produces far greater quantities of chloroplastmonosomes than does the corresponding enzyme from healthy leaves.(ii) The acid soluble oligonucleotide fragments produced bythe soluble RNase from healthy and inoculated leaves (at 48hr after inoculation) in the RNA of isolated ribosomes are quantitativelysignificantly different. This suggests a change in the propertiesof the soluble RNase during the initial stages of host-parasiteinteractions. (iii) As early as 24 hr after inoculation, thereis a dramatic change in the distribution of the pH 5 insolubleand soluble RNase cleavage sites in the RNA of ribosomes indicatinga readily detectable conformational change in the ribonucleoproteinparticles. (iv) These changes in the RNases and ribosomes areonly detectable in the susceptible cultivars of barley and notin a cultivar which is genetically resistant to race 3 of thepowdery mildew fungus. (Received January 31, 1980; )     

Anatomy, Growth and Survival of a Long-hypocotyl Mutant of Cucumis sativus Deficient in Phytochrome B

Plant dry matter (DM) partitioning, survival rates, stem anatomy,and stem water conductivity were investigated in wild-type (WT)and long-hypocotyl (lh) mutant seedlings of cucumber (Cucumissativus) grown as isolated individuals under natural radiation.The lh mutant is severely deficient in phytochrome B. Wild-typeseedlings accumulated more DM than lh seedlings over a 4-weekgrowth period in the glasshouse. Leaf and root DM were higherin the WT but stem DM was higher in the lh mutant. Stem DM perunit length was larger in WT than in lh mutant seedlings, evenwhen the two genotypes were compared at equal whole plant DM,which was achieved by growing the plants under different irradiance.In WT seedlings, the hypocotyl was shorter but thicker, withlarger average cell diameter than the lh mutant. In hypocotyltransverse sections the area occupied by load-bearing tissues(xylem and phloem fibres) and the number and diameter of xylemvessels were larger in WT than lh seedlings. Survival ratesof the lh mutant were normal in the glasshouse but very lowoutdoors due to hypocotyl fracture. The water conductivity ofhypocotyl sections was higher in WT than lh seedlings, but nosignificant differences in water conductivity were observedwhen the root remained attached to the hypocotyl. These resultssuggest (a) that compared to the WT, tall and slender lh plantsare more susceptible to mechanical stresses created by windimpact, and (b) that if the lh lesion affects the phyB geneonly, phytochrome B plays a role in the elicitation of anatomicaland morphological changes that specifically increase fitnessin open environments.Copyright 1994, 1999 Academic Press Cucumis sativus (cucumber), light phenotypes, phytochrome, photomorphogenesis, shade phenotypes, stem growth     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Induction of Phenylalanine Ammonia-Lyase Activation and Isoflavone Glucoside Accumulation in Suspension-Cultured Cells of Red Bean, Vigna angularis, by Phytoalexin Elicitors, Vanadate, and Elevation of Medium pH

Treatment of suspension-cultured cells of red bean, Vigna angularis,with nigeran resulted in an accumulation of isoflavone glucosides,such as daidzein 7-O-ß-D-glucoside, daidzein 7,4'-di-O-ß-D-glucoside,and 2'-hydroxydaidzein 7,4'-di-O-ß-D-glucoside, whichwas accompanied by a transient increase in the activity of phenylalanineanimonia-lyase (PAL). Similar effects were also seen with otherphytoalexin elicitors, such as RNase A and cell wall componentsof Phytophthora megasperma var. sojae. Interestingly, the accumulation of isoflavone glucosides andthe transient increase in PAL activity were induced also byvanadate, a specific inhibitor of plasma membrane adenosinetriphosphatase. K₃PO₄ showed similar effects, but this was ascribedto the elevation of medium pH caused by adding this basic salt.In fact, merely raising the pH of the medium was found to besufficient for the induction of PAL activity. Experiments usinginhibitors showed that the induction depends on RNA and proteinsyntheses. The results are discussed in relation to the possiblemechanism of action of phytoalexin elicitors. ¹ Present address: Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Furocho, Chikusa, Nagoya 464, Japan.     

Biochemical Changes in Nodules of Vigna mungo (L.) During Vegetative and Reproductive Stages of Plant Growth in the Field

Nitrogenase activity in root nodules of Vigna mungo L. attaineda peak at the flowering stage and declined thereafter. Levelsof soluble proteins, particularly leghaemoglobin, and ratesof protein and RNA synthesis declined with nitrogenase. Activitiesof protease and RNase increased with the ageing or nodules.Carbohydrate utilization, sugar levels and ATP were maximumat the early pod stage and gradually declined with age. Theseinterrelated changes point to a loss of nitrogenase activityas the first indicator of nodule senescence that is linked withflowering. Later, losses of proteins, total sugar and ATP wererelated to increased RNase and protease activity and decreasedhexokinase and to a loss in capacity to incorporate amino acidinto protein.Copyright 1993, 1999 Academic Press Vigna mungo (L.), senescene, nitrogenase, leghaemoglobin, field experiments     

Effect of abscisic acid and auxin on ribonuclease during ageing of bean endocarp tissue sections

The activity of RNase increases rapidly upon cutting sectionsof bean (Phascolus vulgaris L. var. Kentucky Wonder) endocarp,peaks within 4 to 8 hr and then declines. This rapid developmentof RNase activity is inhibited by cycloheximide. Auxin (naphthaleneaceticacid, NAA) accelerates the rate of decline of RNase. Abscisicacid (ABA) enhances the level of RNase between 4 and 24 hr,associated with a decline in RNA, and this effect of ABA isobscured in the presence of auxin. ¹ This work was supported by National Science Foundation Grant(GB-8316) to J. A. Sacher. ² On leave from Laboratorio di Radiobiochimica ed EcofisiologiaVegetale, C. N. R., Roma, (Italy), with a Fellowship supportedby North Atlantic Threaty Organization. ³ Present address: Instituto di Botanica, Universita di Ban,Bari, 70126, Italy. (Received April 1, 1971; )