Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Expression of the alpha subunit of 7S nerve growth factor in the mouse submandibular gland

-NGF is an inactive serine protease that is associated in the mouse submandibular gland with a closely related serine protease, -NGF, and the neurotrophic factor, -NGF. The heterogeneity of purified -NGF has been examined by DEAE-cellulose chromatography and SDS polyacrylamide gel electrophoresis. A possible explanation for the observed heterogeneity is presented. Antibodies have been prepared against -NGF and purified by affinity chromatography so that they do not cross-react with -NGF. This antibody preparation recognizes two very similar proteins in male mouse submandibular gland RNA-directed cell-free translation mixtures. The expression of only one of these forms is regulated by testosterone. Oligonucleotide probes specific for each of the three NGF subunits have been prepared and used for Northern blot analysis of RNA from the mouse submandibular gland. The three subunits were found to be coordinately expressed and each were 30-fold more abundant in male than in female glands.Abbreviations used NGF nerve growth factor - -, -, and -NGF -, -, and -subunits of mouse 7S NGF - PBS phosphate buffered saline - DTT dithiothreitol - PPO 2,5-Diphenyloxazole - DMSO dimethylsulfoxide - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - SSC 0.15M NaCl, 15 mM sodium citrate Supported by USPHS research grant NS19964. This paper is respectfully dedicated to Profs. Eric M. Shooter and Silvio Varon in recognition of their many contributions to our understanding of the structure and function of nerve growth factor.     

‘Leaf tip hooding’ — an induced mutation inN. Tabacum,var. Delcrest

A leaf tip hooded mutant was isolated in the progenies of the irradiated seeds of a commercial tobacco variety, namely Delcrest. The mutant character was determined as monogenic recessive. Along with leaf tip hooding, certain other characters such as increase in size of leaves, number and clasping arrangement of leaves on stem, and delayed flowering, were found to be closely associated and this was attributed to the pleiotropic effect of the leaf tip hooding gene.     

Study of androgenetic performance and molecular characterisation of a set of wheat-rye addition lines

Rye chromosomes of wheat-rye addition lines were successfully identified by means of an RFLP analysis with 30 probes. Our results are in agreement with previous cytological data concerning the identity of lines F (+1R), D (+2R), C (+3R), A (+4R), E (+5R) and B (+7R). Two categories of chromosomal rearrangements have been distinguished, namely: (1) deletions: the current line D possesses a chromosome 2R deleted on its short arm and the line G a chromosome 3R deleted on its long arm; we have also noticed a deletion on the long arm of wheat chromosome 1A in line F61; and (2) evolutionary reciprocal translocations in rye relative to wheat which have been previously mentioned in the literature. The anther culture response of the different lines was studied. A significant difference between FEC 28 and the addition lines was observed for embryo production and plant regeneration. It appears that genes located on S 10 chromosome arm 3RL and on FEC 28 chromosome arm 1AL increase embryo frequency whereas gene(s) located on S 10 chromosome 5R reduce(s) it. Plant regeneration results suggest that genes increasing regeneration ability and green-plant frequency are located on S 10 chromosome 4R. The long arm of chromosome 1A seems to be involved positively in green-plant regeneration whereas chromosomes 1R and 3R limit plant regeneration.     

The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation,stoichiometry, reassembly and topology of individual subunits

Summary Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termed, and, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunit is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termed. A fifth, diffuse component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies of and 8 copies of/ within the 2×6 particles. The: ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura. exists in 2 copies inAstacus andCancer which means that a single dimer- is present in a two-hexamer. This leaves 2 monomeric copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacus - was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (F_ab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridge- is surrounded by two and two subunits forming the central core of the dodecamer. Two additional and two additional subunits form the periphery together with one subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This paper is decicated to Professor Dr. Bernt LinzenPreliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)     

Ultrastructure of the pleopods of the estuarine isopod Cyathura carinata (Crustacea: Isopoda: Anthuridea)

Summary The exopodal branches of the pleopods of Cyathura carinata have a thicker cuticle than the endopodites, but the hypodermis proved to be very thin and probably of respiratory function. The nuclei are found in pilaster areas together with stabilizing elements (micro-tubuli attached to cuticular pegs), which are typical for hypodermal tendon cells of other arthropods. Precipitation of Ag₂S revealed that the cuticle of the exopod is more permeable to silver ions than the cuticle of the endopods. The endopodal epithelia have the features of typical osmoregulating tissues (apical folds, mitochondrial pumps, and basal invaginations of the plasma membrane) and reduce silver nitrate. The stabilizing pilaster areas are composed of the same elements as in the exopods.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Structural and functional properties of proteasome activator PA28

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28 and PA28. The purified activator protein (_200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an _{3 3} stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated and subunits, yielding two different oligomers: with the single subunit, PA28 homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated -subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing. Abbreviations: IFN – interferon; LMP – low molecular weight peptide; MHC – major histocompatibility complex.     

An explication of ‘wind illness’ in Northern Thailand

Wind illness is a very common complaint among the Northern Thai, yet is rarely recognized by Thai physicians trained in biomedicine. Persons most susceptible to wind illness are adult women who have ever borne a child. Consequently, data were obtained from 415 everparous women, 43% of whom reported ever having had wind illness and 57%, never having had it. In addition, 20 individuals who had ever had the syndrome were followed for case study, and 13 indigenous healers who traditionally treat clients suffering wind illness were interviewed. Their perceptions of the etiology, symptomatology and treatment of wind illness are reported in Part I. Part II is an attempt to define wind illness in terms of biomedicine and as a consequence of fertility. Part III synthesizes the emic and etic accounts with explanations for the perdurance of wind illness despite the advances of biomedicine and the recent fertility decline in Northern Thailand.     

Composition and mean residence time of molecular weight fractions of organic matter extracted from two soils under different forest species

The organic matter extracted from various mineral horizons of two forest soils, one under silver fir (Abies alba Mill.), the other under European beech (Fagus sylvatica L.), was fractionated by dialysis into three fractions, 100–1000, 1000–8000, and >8000Da. On a C basis, in all horizons the recovered organic matter amounted to less than a half of the total and was mainly composed of molecules >8000Da. The 100–1000Da fraction had a principal elemental composition profoundly different from the other two fractions, which, instead differed from each other significantly only for the S content and the molar ratio of C with N. No significant difference in this regard was found between soils. The richness in O and some typical absorption bands in the FT-IR spectra indicated that the 100–1000Da fraction had a lot of carboxyl moieties. The spectroscopic (¹³C NMR) investigation showed that the 1000–8000 and >8000Da fractions had a prevalently aliphatic nature and signals attributable to polysaccharides (O-alkyl C) revealed overall a high presence of non-humic biopolymers. These latter were significantly more abundant, suggesting a lower degree of humification, in the >8000Da fraction than in the 1000–8000Da fraction. Comparing soils, that under beech appeared significantly richer in O-alkyl C than that under fir. The organics extracted from the A horizon of both soils had positive ¹⁴C values, indicating recent synthesis mainly due to the present forest cover. The mean residence time (MRT) of the combined 100–1000Da and 1000–8000Da fractions and the >8000Da fraction increased with depth, even to about 5000 years in the more than 1-m deep BC horizons under beech. In some cases, and especially in the soil under fir, despite higher values of ¹³C denoting stronger microbial decomposition, the 100–8000Da fraction showed a higher MRT than that of the >8000Da fraction, perhaps due to its ascertained lower content of non-humic biopolymers.     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Self-consistent 3J coupling analysis for the joint calibration of Karplus coefficients and evaluation of torsion angles

The concept of self-consistent J coupling evaluation exploits redundant structure information inherent in large sets of 3J coupling constants. Application to the protein Desulfovibrio vulgaris flavodoxin demonstrates the simultaneous refinement of torsion-angle values and related Karplus coefficients. The experimental basis includes quantitative coupling constants related to the polypeptide backbone torsion originating from a variety of heteronuclear 2D and 3D NMR correlation experiments, totalling 124 3J(HN,H), 129 3J(HN,C), 121 3J(HN,C), 128 3J(Ci–1,Hi), 121 3J(Ci–1,Ci), and 122 3J(Ci–1,Ci). Without prior knowledge from either X-ray crystallography or NMR data, such as NOE distance constraints, accurate dihedral angles are specified for 122 non-glycine and non-proline residues out of a total of 147 amino acids. Different models of molecular internal mobility are considered. The Karplus coefficients obtained are applicable to the conformational analysis of torsions in other polypeptides.     

The biogeochemical cycle of the adsorbed template II: Selective adsorption of mononucleotides on adsorbed polynucleotide templates

Experimental results are presented for the verification of the specific interaction step of the adsorbed template biogeochemical cycle, a simple model for a primitive prebiotic replication system. The experimental system consisted of gypsum as the mineral to which an oligonucleotide template attaches (Poly-C or Poly-U) and 5-AMP, 5-GMP, 5-CMP and 5-UMP as the interacting biomonomers. When Poly-C or Poly-U were used as adsorbed templates, 5-GMP and 5-AMP, respectively were observed to be the most strongly adsorbed species. Moreover, there exists a direct quantitative relationship between the quantity of cytidine or uracil residues in the adsorbed state and the amount of the complementary mononucleotide that is attached to it. NaCl added to the system in order to create conditions of high ionic strength seems to enhance the selectivity of the adsorption of the monmucleotides to these adsorbed templates.     

The chloroplast ATP synthase: Structural changes during catalysis

This article summarizes some of the evidence for the existence of light-driven structural changes in the and subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of the subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modifyLys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the subunit; and (3) release/ exchange of tightly bound ADP from the enzyme. These and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.     

A dominant mutation for high oleic acid content in sunflower (Helianthus annuus L.) seed oil is genetically linked to a single oleate-desaturase RFLP locus

Pervenets is a sunflower mutant with a seed oil oleic acid content greater than 65%. It was obtained after mutagenesis treatment on VNIIMK 8931. Several commercial varieties derived from Pervenets and breeding materials with a high oleic acid content have been marketed. However, the genetics of this trait are still not fully understood by breeders. To characterize the Pervenets mutation, we studied RFLP in relation to high oleic acid content. We performed diversity analyses on 239 genotypes with cDNA sequences coding for 9- and 12-desaturases as probes. The 12 RFLPs enabled us to identify at least two independent loci. One 12 RFLP allele (12HOS) was strictly correlated to high oleic acid content, whereas no correlation was found between 9-desaturase polymorphism and high oleic acid content. These results enabled to us estimate the genetic distance between the marker and the Pervenets mutation loci. An F₂ segregating population of 107 plants confirmed the correlation between high oleic acid content and 12HOS, indicating tight genetic linkage. The nature of the Pervenets dominant mutation and the complexity of the high oleic acid content trait are discussed.     

Activation of fatty acids by non-glyoxysomal peroxisomes

Ethylene treatment (approx. 20 l ·1^-1 in air for 2 d) of tobacco (Nicotiana tabacum L. cv. Havana 425) plants markedly increases the endo--1,3-glucanase (EC 3.2.1.39) content of leaves. The antigenic form of the enzyme induced is the same one whose production is blocked by treating cultured cells with combinations of auxin (1.1 · 10^-5 M -naphthaleneacetic acid) and cytokinin (1.4 · 10^-6 M kinetin). Evidence is presented that cultured tobacco cells require ethylene for -1,3-glucanase accumulation: i) ethylene treatment increased the accumulation of \-1,3-glucanase in callus tissues >10 d after subculturing and in cell-suspension cultures; ii) callus tissues can produce ethylene; iii) conditions known to inhibit ethylene production (1 mM CoCl₂; 33° C treatment) or ethylene action (approx. 1.6 mmol · 1^-1 norbornadiene in air) inhibited -1,3-glucanase accumulation by callus tissues treated for 4 d following subculturing; and, these inhibitory effects were prevented by exogenous ethylene. Combinations of auxin and cytokinin blocked ethylene-induced accumulation of -1,3-glucanase by cell-suspension cultures. The results favor a model in which ethylene induces results favor a model in which ethylene induces 1,3-glucanase accumulation, and auxin and cytokinin inhibit this induction process.Abbreviations NAA -naphthaleneacetic acid - NDE norbornadiene