1,721,083 research outputs found
Effects of EGTA on the increase in Calmodulin level in early germination of radish (Raphanus sativus) seeds
No full textEFFECTS OF EGTA ON THE INCREASE IN CALMODULIN LEVEL IN EARLY GERMINATION OF RADISH (RAPHANUS SATWUS) SEEDS.
M. Cocucci and N. Negrini - Istituto di Chimica agraria, Universita di Milano, via Celoria 2 Milano.
In a previous paper we have shown that the metabolic reactivation which takes place durm& germination of radish seeds (Raphanus sativus) is accompanied by an mcrease in CaImodulin (CaM) level (+450 % at 24h). The presence of a proteinaceous CaM inhibitor m ungerrninated seeds was shown and a strong reduction in its level during metabolic reactivation was re~rted. In the present study the effect of CaZ+ chelator EGTA on metabolic reactivation and increase in CaM level is reported. The presence of EGTA in the incubatIon medium inhibited germination, measured as increase in fresh weight of embryo axes, at concentrations higher than 1 mM and at 5 mM the increase in fresh weight at 24h was inhibiteiI by ca. 50%. Incubations in 1 and 5 mM EGTA reduced Ca2+ content Qf embryo axes at 24h incubation by ca. 30 and 35 % respectively. CaZ+ in the incubation medium was very low in the control. In the medIUm of the seeds incubated in the presence of EGTA the level of Ca2+ was high but the free Ca2+ was very low. Metabolic reactivation, measured as increase in RNA and DNA contents in embryo axes, was not affected by 1 mM EGTA but was slightly reduced by 5 mM EGTA; a toxic action of 5 mM EGTA was also shown by a large leakage of K+ in the incubation medium. When the seeds were incubated m the presence of EGTA and Ca2+ at the same concentration the germination and metabolic reactivation were very similar to the con!(olindi.cat~g that the e.ffect of EGTA dependend on the decrease in CaZ+availabIlity. The increase in CaM level in the soluble fraction of embryo axes incubated in the presence of 1 mM EGT A was inhibited by 50% and its specific activity was reduced in a similar way. When the soluble fraction was analyzed on a
Sephadex G 75 SF column, CaM was eluted in several peaks in tlie zone at MW higher than 30 kD. In seeds incubated in the presence of EGTA there was a reduction of CaM eluted in the zone at lower MW . In such zone CaM was eluted with the inhibitor. The fact that at 1 mM EGTA in the incubation medium there was only a slight decrease in germination and metabolic reactivation even if CaM level was 50% lower suggests that Ca2+ might control the increase in CaM level which takes place in early germination and that in this same period the role of CaM could lie also in relatIon with its Ca2+ chelating capacity. The control of the activities dependent on Ca-CaM could be also in relation with the presence in the seeds of the proteinaceous CaM inhibitor
Calmodulin levels in radish (Raphanus sativus L.) seeds germinating at low calcium availability, induced by EGTA treatments
No full textIncubation of radish (Raphanus sativus L.) seeds in the presence of 1 or 5 mol m(-3) Ca-EGTA, which increased Ca2+ activity in the incubation medium (c. 0.24 or 0.37 mol m-3 at 24 h with respect to c. 0.13 mol m(-3) in the control), did not affect germination, the restoration of K+ net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m(-3) Na-EGTA, which reduced Ca2+ activity in the incubation medium (20 mmol m(-3) at 24 h), decreased the total Ca2+ level in embryo axes (-21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K+ net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m(-3) Na-EGTA (Ca2+ activity in the incubation medium was 0.6 mmol m(-3)), the decrease in the total Ca2+ level was greater (c. -27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca2+ availability does not affect germination and suggest that the effect of Na-EGTA, at least up to 5 mol m(-3), is a result of an induction of Ca2+ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000 g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5-fold and 7-fold, respectively). This increase was very similar in the Ca-EGTA-treated seeds but was inhibited (c. -38%) by 1 mol m(-3) Na-EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m(-3) Na-EGTA (c. -30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci and N. Negrini (1988) Plant Physiology 88, 910-914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca2+ availability and that the CaM inhibitor has a role in controlling the amount of Ca-CaM available for the Ca-CaM-dependent enzymes
Evaluation of the changes in biochemical and physiologica parameters as indicators of heavy metal pollution
No full textEVALUATION OF THE CHANGES IN BIOCHEMICAL AND PHYSIOLOGICAL
PARAMETERS AS INDICATORS OF HEAVY METAL POLLUTION
A. Rivetta, N. Negrini, M. Cocucci
Department of Plant Physiology and soili chemistry, University of
Milan, Milan Italy
The lIse of biological system, particularly plants, could be an useful tool in monitoring polluted environments. It is known that plants incubated in the prc ,,:,cllce of heavy metals can accumulate thiels, low molecular weight molecules rich in cysteine and can
change the level and the express ion of many enzymatic activit ies. [n this work wc consider the possibility to use the changes in the !evel of acid-soluble thio ts, as
physiological markers of heavy metal pollution. [nroots and shoots of two different spec ies, the level of acid-solu ble th iols earl y and strongly increased when incubated in the presence of Cd2+. Thiols level also increased in plants grown on a soil-like standard substrate supplied with different amounts of Cd2+ or with increasing amounts of a heavy metal po[(utcd soil up to 10% (dry wt:dry \Vt). In both cases, when Cd2 in plant
shoots reached the same level (30 nmol g [ fw), the increase in thiols level was higher in plants grown in the presence of polluted soil than Cd2+. Anyway, the increase in the level of thiols was not accompanied by the inhibition of plant growth, as indicated by the 51igh changes in fresh weight. protein and chlorophy ll content, with respect to the control. These data suggest that the changes in the level of thiols may be considered a
physiological parameter indicator of the presence of heavy merals in the soil and may be used also in field conditions. Moreover, using glutathione affinity chromatography, preliminary Jula suggest that changes in glutathione-dependent activ it ies , in particular
glutathione transferase. may be a very sensible marker of Cd2+ stress
Biosynthetic activities are not involved in germination and metabolic seed reactivation promoted by fusicoccin
No full textChanges in the levels of Calmodulin and of a Calmodulin inhibitor in the early phases of radish (Raphanus Sativus) seed germination : effects of ABA and Fusicoccin
Full text linkEffect of high osmotic pressure on cam levels in germinating radish (Raphanus sativus) seeds
No full textPossibile coinvolgimento del Ca2+ nel contrastare l’effetto di alte concentrazioni di NaCl durante la germinazione del seme di ravanello
No full textPOSSIBILE COINVOLGIMENTO DEL Ca2+ NEL CONTRASTARE L'EFFETTO DI
ALTE CONCENTRAZIONI DI NaCI DURANTE LA GERMINAZIONE DI SEMI DI
RAVANELLO
M. Cocucci, N. Negrini, A. Spinardi
Dip. di Fisiologia delle Piante Coltivate e Chimica Agraria, Università degli Studi di Milano.
Riassunto
Lo stress salino, che compromette gravemente la crescita delle piante, è caratterizzato da una
componente di toss icità del Na+ ed una asmatica, gli effetti delle quali sono difficilmente
scindibili. Durante tale stress il Na+ viene assorbito rapidamente dalle cellule ed è noto il suo
effetto nell'interferire a vari livelli col Ca2+ E' apparso quindi interessante studiare l'effetto di tale
stress e delle sue componenti durante la riattivazione metabolica che caratterizza le prime fasi di
germinazione del seme di ravanello (Raphanus sativus), processo nel quale l'omeostasi del Ca2+
deve essere strettamente controllata e potrebbe essere alterata dalla presenza del Na+
La presenza di crescenti concentrazioni di NaCl (da 50 a 300 mM) nel mezzo di incubazione
inibisce fortemente e progressivamente la crescita, misurata come incremento di peso fresco, di
assi embrionali di semi di ravanello durante le prime 24 h di germinazione. Parallelamente a tale
inibizione si osserva una progressiva riduzi one della capacità dei semi di riassorbire K+ dal mezzo
esterno accompagnata da un aumento del rilascio del Ca2+ L'analisi dei contenuti ioni ci degli assi
embrionali dei semi germinati 24 h in tali condizioni indica una progressiva riduzione dei livelli di
K+ ed un forte aumento dei livelli di Na+, ri spetto al controllo germinato in acqua. I livelli interni
di Ca2+ non subiscono variazioni. Nell'intento di valutare se l'azione del NaCl sulla crescita e i
contenuti ionici degli assi embrionali fosse acrivibile al Na+ e non piuttosto al Cl - o ad un effetto
asmatico, dovuto alle alte concentrazioni saline, si è ana lizzata la variazione dei suddetti parametri
in presenza di concentrazioni equimolari di Na+ sotto forma di S042- ed equiosmolari di mannite.
Per quanto riguarda il Na2S04 i suoi effetti sono simi li a quelli osservati in presenza di NaCI. La
mannite, a tutte le concentrazioni saggiate, riduce solo del 30% l'incremento di peso fresco degli
assi embrionali, rispetto al controllo germinato in acqua, mostrando un'inibizione simile a quella
indotta da NaCl 50 mM. Non si osserva alcun effetto della mannite sulla riassorbimento di K+ dal
mezzo di incubazione e sui livelli di K+ e Ca2+ negli assi embrionali. In tali condizioni risulta
tuttavia inibito il rilascio di Ca2+ che ha luogo durante le prime 24 h di germinazione. Questi dati
suggeriscono che l'azione del NaCl durante la germinazione del seme di ravanello sia da ascriversi
ad un effetto dovuto in particolare al Na+, e solo in minima parte ad un effetto asmatico del sale.
Il Ca2+ sembra essere in qualche modo coinvolto nella regolazione di tali effetti.
Il poss ibile ruolo del catione è stato analizzato somministrando ai semi germinanti concentrazioni
crescenti di NaCl in assenza e in presenza di CaCl2 10-3 M. La presenza del Ca2+ nel mezzo di
crescita riduce significativamente l'effetto inibente del Na+ sull'incremento di peso fresco degli
assi embrionali. Esso favorisce inoltre un più efficiente riassorbimento del K+ dal mezzo da parte
dei sem i trattati con NaCI. Parallelamente negli assi embrionali di semi trattati con NaCl in
presenza di Ca2+ non si osserva riduzione dei livelli di K+, se non alle più alte concentrazioni
saline somministrate, e l'incremento dei livelli di a+ risulta fortemente ridotto (ca. -30%) rispetto
ai controlli in sa le. In queste condizioni i livelli di Ca2+ interno non variano. Misure del potenziale
elettrico transmembrana in cellule di assi embrionali di semi genuinati in condizioni di controllo o
in mezzo contenente NaCl 100 mM, in assenza e in presenza di CaCl2 10-3 M, indicano un
significativo effetto del Ca2+ nel ridurre la forte depolarizzazione indotta dal Na+
I dati otten uti suggeriscono un ruolo del Ca2+ nel contrastare gli effetti dello stress salino
indicando un'azione protettiva dello ione dagli effetti tossici del a+
10
Calmodulin binding sites on microsomal fraction of germinating radish (Raphanus sativus) seeds
No full text
- …
