Carrot cells

A more general role of ABA in stress tolerance has been found in carrot cells. When a suspension culture of carrot cells was exposed to ABA and then selected for tolerance to freezing, the ABA-treated cells were found to be more tolerant to the stress (Reaney Gusta, 1987). These results provide further evidence for the presence of common mechanisms conveying tolerance to many of the environmental stresses. 

Reuveni, M., Colombo, R., Lerner, H.-R., Pradet, A. Polkajoff-Mayber, A. (1987). Osmotically induced proton extrusion from carrot cells in suspension culture. Plant Physiology, 85, 383-8. 

Fig. 10. Intercellular junction zones of carrot cells grown in suspension have been observed in electron microscopy after immunogold labeling with the 2F4 antibody, (a) no treatment of the sections prior to labeling the gold particles are restricted to the center of the junction zones (b) enzymatic (pectin methyl esterase) deesterification of the E.M. grids before labeling the deesterified pectins present in the primary walls now bind the probe. Scale bars = 1 pm.

Fig. 11. The PAL enzymatic activity of carrot cells has been measured in the presence of pectic fragments of DP>9 (a) controls and treatments have been performed in solutions with varying Ca " to Na" ratios (b) the PAL activity is expressed as a function of the recognition by 2F4 antibodies of the pectic fragments incubated in the same salts solutions as in (a).

Fig. 12. Tentative model of the signal transduction chain that links the perception of pectic fragments to defense responses in carrot cells. Abbreviations apy, heterotrimeric G protein CaM, calmodulin 4CL, 4-coumarate-CoA ligase CTX, cholera toxin FC, fusicoccine GDP-P-S and GTP-y-S, guanosine 5 -0-(2-thiodiphosphate) and guanosine 5 -0-(3-thiotriphosphate) IP3, 1,4,5-inositol trisphosphate PAL, phenylalanine ammonia-lyase PLC, phospholipase C PR, pathogenesis related PTX, pertussis toxin Rc, receptor SP, staurosporine. Activation and inhibition are symbolized by + and -respectively.

The third described enzyme form with pH optimum about 4.7 [11, 4], we found in Fraction C - the fraction from carrot roots pulp (Fig. 2). We supposed that this form of exopolygalacturonase is relatively strongly bound on carrot cell walls and so it can be released only by higher salt concentrations. The approximative molecular mass determination on Superose 12 (Fig. 3c) showed the molecular mass about 50 000 for this form and the second, with more acidic pH optimum, form present in the fraction. The further characterization of these enzymes showed the exopolygalacturonase with pH optimum 4.7 to be identical with enzyme described sooner by Pressey and Avants [4] and exopolygalacturonase with pH optimum 3.8 to be identical with the enzyme from Fraction A. In conclusion, the exopolygalacturonase form with pH optimum 3.8 can be considered to be the main enzyme form present in carrot roots. 

Kang YH, Parker CC, Smith AC and Waldron KW. 2008. Characterization and distribution of phenolics in carrot cell walls. J Agric Food Chem 56(18) 8558-8564... 

KUROSAKI, F KIZAWA, Y., NISHI, A., Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts, Eur. J. Biochem., 1989,185, 85-89. 

DC034 Sugano, T., T. Tanaka, E. Yamamoto, and A. Nishi. Behaviour of phenylalanine ammonia-lyase in carrot cells in suspension cultures. Phytochemistry 1975 14 2435-2436. 

Physiological changes of carrot cells in suspension culture during growth and senescence. Physiol Plant 1975 33 251. 

DC085 Han, A., K. P. Zanewich, S. B. Rood, and D. K. Dougall. Gibberellic acid decreases anthocyanin accumulation DC095 in wild carrot cell suspension cultures but does not alter 3 nucleosidase activity. Physiol Plant 1994 92(1) ... 

Bar Nun. Identification of the major anthocyanin of carrot cells in tissue DC135 culture as cyanidin 3 (sinapoylxylo sylglucosylgalactoside). Z Naturforsch Ser 1983 C38(ll/12) 1055-1056. 

Salem, S., D. Linstedt, and]. Reinert. The cytokinins of cultured carrot cells. Protoplasma 1979 101 526-534. Sasse, F., D. Backs-Husemann, and W. Barz. Isolation and characterization of vacuoles from cell suspension cultures of Daucus carota. Z Naturforsch 1979 Ser C 34 103-109. 

Moriya, and A. Nishi. Increase in enzyme levels during the formation of phenolic acids in carrot cell cultures. DC 180 Phytochemistry 1978 17 1235-1237. 

DC 193 Kurosaki, E., and A. Nishi. Isolation and antimicrobial activity of the phytoalexin 6-methoxymellein from cul- DC205 tured carrot cells. Phytochemistry 1983 22(3) 669-672. 

Takeda, J. and Abe, S., Light-induced S5mthesis of anthocyanin in carrot cells in suspension. IV. The action spectrum, Photochem. Photobiol, 56, 69, 1992. 

Liners, F., Van Cutsem, P. (1992). Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells - An immunocytochemical study. Protoplasma, 170,10-21. 

Relton, J.M. Bonner, P.L.R. Wallsgrove, R.M. Lea, P.J. Physical and kinetic properties of lysine-sensitive aspartate kinase purified from carrot cell suspension culture. Biochim. Biophys. Acta, 953, 48-60 (1988)... 

However, little is currently known about its synthesis. The protein component may be assembled on the ribosomes by the normal mechanism of protein synthesis.324 L-Proline is known to be the precursor of the hydroxy-L-proline found in the glycoprotein,324,325 hydroxylation of the peptide-bound L-proline being catalyzed, in carrot cells, by cytoplasmic enzymes.324... 

INDUCTION AND REGULATION OF BIOSYNTHETIC ACTIVITY OF PHYTOALEXIN IN CARROT CELLS... 

Further support for the hypothesis that Ca2+ plays a central role in regulating phytoalexin accumulation is provided by experiments in which the turnover of phosphatidylinositol was measured in the plasma membrane of elicitor-treated carrot cells [17]. The carrot cells were first labelled with [3H]myo-inositol and, after the addition of elicitors, acid extracts of the cells were analyzed chromatographically for the production of inositol trisphosphate (IP3). In cells treated with elicitor, the release of radioactive IP3 increased with time and attained a maximum at 3 - 5 min after treatment. Phospholipase activity responsible for the degradation of phosphorylated phosphatidylinositol increased correspondingly. Several reports have shown that IP3 induces rapid release of Ca2+ from intracellular stores in animal cells [18, 19]. Studies on plant cells have also demonstrated that exogenous IP3 releases Ca2+ from microsomal preparations at micromolar concentrations, although only limited... 

Constitutive activity of PDE was found in cultured carrot cells this activity did not depend on either Ca2+ or CAM. By contrast, a CAM-dependent isoform of PDE (CAM-PDE) was induced in the cells by adding forskolin or Bt2cAMP to the culture [30]. Induction of CAM-PDE activity in Bt2cAMP-treated carrot cells was markedly inhibited in the presence of verapamil, and addition of Ca2+-ionophore A23187 induced CAM-PDE [34]. These results suggest that increased Ca2+, but not cAMP, in the stimulated carrot cells triggers induction of the PDE isoenzyme. Affinity of CAM-PDE to the substrate was low compared to constitutive PDE Km values, 0.14 and 0.07 pM, respectively) however, V for the induced PDE was approximately 2.7 times higher than for the constitutive isoenzyme. 

These results suggest that synthesis and degradation of cAMP in cultured carrot cells are both controlled and switched on/off according to the concentration of Ca2+ in carrot cytoplasm. Adenylate cyclase activity is induced in the cells only in the resting state, and the enzyme activity is... 

The biochemical basis of CAM-induced stimulation of Ca2+-ATPase activity in carrot cells was studied further by determining the parameters of the Ca2+-translocating reaction of the enzyme in the presence and absence of exogenous CAM, using EGTA-treated plasma membrane [45], The affinity of Ca2+-ATPase for Ca2+ was considerably increased by... 

Since preliminary studies showed that 6-hydroxymellein-O-methyl-transferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that S-adenosyl-Z.-homocysteine (SAH), which is a common product of many O-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-Omethyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the specific product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6-methoxymellein inhibits transferase activity with respect not only to 6-hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein-O-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6-methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should... 

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