Significant role for polysomes associated with the cytoskeleton in the control of protein synthesis during germination of triticale caryopses in the presence of abscisic acid

The influence of abscisic acid (ABA) on the process of polysome formation and synthesis of newly-formed proteins by different polysome populations was studied. Triticale caryopses were germinated in water or various ABA concentrations for 48 hrs, and afterwards they were transferred to a solution of ¹⁴C-amino acids and germinated for an additional 30 min. Embryos were separated from caryopses, and four polysome populations were isolated: the FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). ABA retarded both the process of polysome formation and their activity in forming new proteins in vivo in all studied fractions. Participation of polysomes in total ribosomal materials (sub-units, monosomes and polysomes) of each polysome population in the control sample was as follows: FP — 77; MBP — 72; CBP — 70 and CMBP — 66 %, whereas in sample treated by ABA (100 μM) it was accordingly: 17; 23; 27 and 28%. The largest population made up FP (in control sample 69%), participation of MBP was always lower and ranged from about 19 to 30 %. Participation of polysome populations bound with the cytoskeleton CBP and CMBP, both in control sample as well as in samples treated with 1 and 10 μM ABA solution, was only a few per cent. It should be noted that when the ABA concentration was higher (100 μM) (process of germination was strongly inhibited), participation of those two populations (CBP and CMBP) was much increased in embryos, respectively to about 18 and 20 %. In both the control group and in embryonal tissue treated with ABA increasing incorporation of radioactive precursors to newly-formed proteins in vivo in fractions of polysomes isolated by following buffers: C (FP), C + PTE (MBP), C + Tris (CBP) and buf. U (CMBP) was observed. It should be noted, that the biggest incorporation of ¹⁴C-amino acids into nascent polypeptide chains was found in the last polysome population (CMBP). In the sample treated with ABA (100 μM) the activity of this fraction (CMBP) in forming new proteins is several times, and in the case of FP dozens of times, more intense. Increased participation of CBP and CMBP in embryos of triticale caryopses treated with ABA (100 μM) and the largest incorporation of ¹⁴C-amino acids into nascent polypeptide chains synthesised by CMBP, may indicate the important role of proteins formed by polysomes associated with cytoskeleton in inhibition of germination and seedling growth by ABA.     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 μM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.     

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.     

Effect of osmotic stress on the formation of a population of polysomes and their stability in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) seeds

Plants growing under natural conditions are constantly exposed to various stress factors, which can restrain their productivity and limit yields. This paper deals with the effect of long- and short-term osmotic stress followed by recovery on the formation of polysomes and their stability during germination of pea (Pisum sativum L.) seeds. By isolating polysomes, it is possible to obtain an index which evidences the ability of tissues to synthesize proteins. Changes in the distribution of polysomes often precede measurable changes in amounts of proteins. Under osmotic stress, the dominant population of polysomes was the population of free polysomes (FP). The share of membrane-bound polysomes (MBP) and cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP) in the total fraction of ribosomes increased under intensive (−1.0 and −1.5 MPa) osmotic stress. These results can suggest that the bound forms of polysomes play an important role in the synthesis of stress proteins. In addition, the stability of polysomes isolated from pea early seedlings growing under unstressed control and osmotic stress conditions was tested. It turned out that polysomes formed under osmotic stress conditions (especially the CMBP) were more resistant to the activity of exogenous ribonucleases than the polysomes in the control samples. Under stress conditions it is highly likely that ribosomes become more densely packed on mRNA thus making it more resistant to ribonuclease. This is just one of the many mechanisms regulating stability of mRNA.     

Morphological changes in Krebs II ascites tumour cells induced by insulin are associated with differences in protein composition and altered amounts of free,cytoskeletal-bound and membrane-bound polysomes

A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183–193,1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture- Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more then 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins. (Mol Cell Biochem 118: 131–140, 1992)     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane     

The effect of insulin on proteins associated with free,cytoskeletal-bound and membrane-bound polysome populations.

It is known that insulin treatment increases the rate of protein synthesis in many cells and tissues and that it causes changes in the distribution of ribosomes between free (FP), cytoskeletal-bound (CBP) and membrane-bound polysome (MBP) populations. This paper concerns an analysis of the pattern of proteins in high-salt extracts of FP, CBP and MBP isolated from Krebs II ascites and MPC-11 cells. A combined detergent/salt extraction procedure was used to isolate the three fractions of polysomes from control cells and from cells following short-term stimulation with insulin. There were differences in the protein patterns in the individual fractions and changes occurred after insulin stimulation.     

Free,cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins

A three-step detergent/salt extraction procedure (Vedeleret al., Mol Cell Biochem 100: 183–193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins. The results show differences in the poly(A) binding properties of the proteins in the three polysome fractions and that the complements of polysome-associated proteins are different in the two cell lines. This may be related to the differences in the growth characteristics of MPC-11 and Krebs II ascites cells.     

Exogenous abscisic acid increases stability of polysomes in embryos of triticale caryopses during germination

Some posttranslational processes that occur in embryos of germinating triticale caryopses treated with different concentrations of abscisic acid (ABA) were examined. ABA increased the ratio of cytoskeleton-bound polysomes in the total population of polysomes and depressed the share of free and membrane-bound polysomes. Using exogenous RNase, stability of the total polysomal population as well as each polysomal fraction was investigated. The total extractable polysomes isolated from embryonic tissues of germinating triticale caryopses treated with ABA were more stable than the polysomes isolated from the control sample caryopses. The contribution of the polysomes that were not digested by RNase was increased by higher concentrations of ABA applied during germination. At high concentrations of ABA (50, 100 μM), the quantitative contribution of polysomes in the total ribosomal fraction was almost 100% of the amount of polysomes before digestion and the modifications observed consisted mainly of the shift of the so-called heavy polysomes towards light polysomes, containing a few ribosomes. Within each polysomal population, cytoskeleton-bound polysomes (CBP and CMBP) were the most stable, which may imply that the bonds between polysomes and these protein filaments, created in all eukaryotic cells increased their stability. It is assumed that mRNAs are stabilised or destabilised by interaction of proteins with their various sequences. A plant hormone may depress or elevate the quantities of these proteins, thus regulating the stability of different mRNAs. The results confirm the multi-faceted mechanism of ABA-induced response, where one of the constituents is the effect of ABA on the stability of mRNAs molecules. The co-ordinated regulation of mRNAs synthesis and their stability provide plants with improved adaptability.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum     

Formation and stability of polysomes and polysomal populations in roots of germinating seeds of soybean (Glycine max L.) under cold,osmotic and combined cold and osmotic stress conditions

Abiotic stress factors such as extreme temperatures or osmotic stress are among the major causes of inferior crop yields. In response to a stress, plants have evolved various defense mechanisms. In our study, we have demonstrated how cold stress, osmotic stress and a combination of both stresses retard the growth of roots and inhibit the process of ribosomes binding into polysomes. The tested stresses also limited the ability of root tissues to synthesize proteins. At the same time, most of the analyzed samples were found to contain elevated shares of the fractions of cytoskeleton-bound polysomes (CBP, CMBP) in the total population of polysomes. Using a polysome-based degradation system, it was shown that polysomes formed under stress conditions were much more resistant to the effect of exogenous ribonuclease than the control ones. The highest tolerance to digestion was demonstrated by the cytoskeleton-bound (CBP) and cytoskeleton-membrane bound polysomes (CMBP). The increasing share of CBP and their stability in roots of seeds germinating under stress conditions can be a target for physiological regulation. It seems that modifications in the stability and percentages of particular polysomal populations play an important role in the adaptation of plants to stress conditions, which may indicate that these forms of polysomes, i.e., cytoskeleton-bound ones, are involved (via selective translation) in the synthesis of stress proteins in soybean roots.     

Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli.

It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes. In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins. Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes. In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned. We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery. It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes. On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

Identity of an ABA-activated 46 kDa mitogen-activated protein kinase from <Emphasis Type="Italic">Zea mays</Emphasis> leaves: partial purification,identification and characterization

Mitogen-activated protein kinase (MAPK) cascades have been shown to be important components in abscisic acid (ABA) signal transduction pathway. In this study, a 46 kDa MAPK (p46MAPK) induced by ABA was partially purified from maize (Zea mays) by Q-Sepharose FF, Phenyl-Sepharose FF, Resource Q, Mono QTM 5/50 GL, poly-l-lysine-agarose, and Superdex 75 prep-grade columns, and was identified as ZmMAPK5 (gi|4239889) by the matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. Furthermore, the kinase showed optimal activity at pH 8.0, 30°C, and 10 mM MgCl₂; the K _m for myelin basic protein (MBP) substrate and ATP were 0.13 μg μl⁻¹ and 62 μM, respectively. MBP was the preferred substrate, of which the threonine residue was phosphorylated. Finally, the kinase was found to respond to diverse extracelluar stimuli. These results enable us to further reveal the function of the ZmMAPK5 in ABA signaling. The authors Haidong Ding and Aying Zhang contributed equally to the work.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

The Percentage of Cytoskeleton-Bound Polysomes as Affected by the Physiological Condition of Plant Cells

The effects of illumination, temperature, deficiency of water or oxygen, phytohormones, and seedling age on the proportion of cytoskeleton-bound polysomes (CBP) in plant tissues were studied. This proportion was shown to vary depending on factors affecting protein synthesis: it decreased under the influence of factors inhibiting protein synthesis and reducing the relative content of polysomes in tissues (oxygen and water deficiencies, seedling aging) and vice versaincreased under the influence of the factors promoting translation (cytokinin, light). The significance of reversible polysome attachment to the cytoskeleton as a mechanism controlling protein synthesis is discussed.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.     

Effects of abscisic acid on content and biosynthesis of terpenoids in Cannabis sativa at vegetative stage

The influence of abscisic acid (ABA) on plastidial and cytosolic terpenoids and on two key enzymes for terpenoid biosynthesis was determined in vegetative stage of Cannabis sativa L. Low concentration of ABA (1 μM) increased 1-deoxy-D-xylulose 5-phosphate synthase (DXS) activity in treated plants in comparison to control plants. The amounts of chlorophyll a and carotenoids increased in response to ABA treatment but chlorophyll b content declined. The accumulation of α-tocopherol was stimulated only by 10 μM ABA. The ABA-treated plants showed a decline in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity which was followed by a decrease in squalene and phytosterol content. ABA also decreased tetrahydrocannabinol (THC) and cannabidiol (CBD) contents. The essential oil had higher ratios of monoterpenes to sesquiterpenes as ABA-treated plants had less numbers of sesquiterpenes in comparison with control plants. Influence of ABA on the amounts of sesquiterpenes was different, some of them showed decrease of content and others increase of content.