Angel Matilla

The relationships between ethylene production and germination ofCicer arietinum seeds

The germination percentage of chick-pea (Cicer arietinum) Seeds was greatly reduced by temperatures of 30°C and 35°C. This thermoinhibition was overcome by ethylene (ethrel). Both ABA and PEG diminished ethylene production and germination percentage in a parallel way. FC, MGBG and CHA stimulated both ethylene production and germination. AVG reduced ethylene production to some extent but did not inhibit germination. CoCl2 and PG completely prevented both ethylene production and germination; this effect was reversed by ethylene but not by its immediate precursor ACC. NBE prevented both germination and ethylene production. Our results suggest that high ethylene production rates are not essential for germination of chick-pea seeds but that certain quantities of ethylene may be required.

Free polyamines in Cicer arietinum seeds during the onset of germination

Abstract Alterations in the content of putrescine (Put), spermidine (Spd), spermine (Spm) and cadaverine (Cd) in the cotyledons and embryonic axis of Cicer arietinum during the first 24 hr of germination have been studied. The embryonic axis attached to the cotyledons contains a larger amount of these polyamines than the cotyledons themselves and larger amounts of Put, Spd and Spm than embryonic axes excised from the whole seeds before germination. Embryotomized cotyledons, on the other hand, show far greater polyamine levels than whole seeds, implying that the cotyledons are at least a partial source of polyamines for the embryonic axis. Nonetheless, Cd is greater in excised embryonic axes, suggesting some control over its synthesis in this organ by the cotyledons. In the embryonic axis, RNA synthesis coincides with an accumulation of Spm and Spd and that of DNA with an increase in Spd and Put. The greatest Spd: Put ratio occurs in the apical meristem of the embryonic axis, whilst Put accumulations are greatest in the elongation zone.

Requirement of polyamines for in-vitro maturation of the mid-binucleate pollen of Nicotiana tabacum

Summary We have studied the changes in endogenous levels of both free and conjugated polyamines (PA) during «in-vitro» transition of pollen grains of Nicotiana tabacum from the mid-binucleate (MB) stage to matured MB (MBM) and matured germinated MB (MBG) stages. Spermidine (Spd) was the most abundant PA in all stages, followed by putrescine (Put) and spermine (Spm). An increase in all forms of PA was observed in the transition of MB to MBM; however, a decrease from MBM to MBG was obtained. The pollen matured «in-vitro» contains more PA than the one matured «in-situ» surrounded by sporophytic tissue. This increase may be due to the fact that glutamine (present in the maturation medium) is related to arginine and ornithine metabolism. The effect of PA biosynthesis inhibitors, DL-difluoromethylarginine (DFMA), DL-difluoromethylornithine (DFMO) or cyclohexylamine (CHA), on maturation and germination processes was also studied. The results suggested that PA biosynthesis «via ornithine decarboxylase» is required for «in-vitro» maturation. However, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) appeared to be implicated in «in-vitro» as well as «in-situ» germination processes. The inhibition induced by DFMO and DFMA was overcome by addition of Put. The content of all forms of PA was also quantified in MB pollen matured in the presence of PA inhibitors in order to support the importance of PA biosynthesis in the maturation process.

Polyamines in the induction of Nicotiana tabacum pollen embryogenesis by starvation

Summary An increase in both putrescine (Put) and spermidine (Spd) bound to macromolecules was observed during «in vitro» embryogenic induction from isolated mid-binucleate (MB) pollen of Nicotiana tabacum . Free polyamines and Spd bound to small substances decreased in this period. We noticed in the fully formed embryos from embryogenic pollen (EP): (1) an increase in free Put and both Put and spermine (Spm) bound to small substances; (2) a decrease in both free Spd and Spm, and in Spd bound to small substances; and (3) a noticeable decrease of polyamines (PA) bound to macromolecules. During the process of embryogenesis, a gradual disappearance of ornithine decarboxylase (ODC) activity was observed. However, arginine decarboxylase (ADC) activity decreased along the induction period, increasing again during the embryo formation. Experiments with the inhibitors of polyamine synthesis difluoromethylornithine (DFMO) and difluoromethylarginine (DFMA) confirmed that PA biosynthesis is required for the embryo formation, but not for embryogenic induction.

Alterations of the ethylene pathway in germinating thermoinhibited chick-pea seeds caused by the inhibition of polyamine biosynthesis

Abstract The alterations in the ethylene biosynthetic pathway provoked by cyclohexylamine (CHA) and/or methylglyoxal-bis(guanylhydrazone) (MGBG), inhibitors of the polyamine synthesis, have been studied in thermoinhibited seeds (delayed germination by supraoptimal temperatures) of the chick-pea (Cicer arietinum). CHA and/or MGBG stimulated ethylene synthesis both at 25°C (control) and at 30°C (temperature at which thermoinhibition is induced, and reversed by both inhibitors). Moreover, both inhibitors caused: (1) an increase in ACC-synthase (EC 4.4.1.14) and free ACC; (2) a stimulation of in vivo ACC-oxidase; and (3) a stimulation of (malonyl)-1-aminocyclopropane-1-carboxylic acid synthesis at 25°C (since the supraoptimal temperatures had no effect). Compared with the control, both supraoptimal temperatures lead to a decrease the AdoMet-decarboxylase (AdoMetDC) (EC 4.1.1.50) activity. This was not affected by CHA. However, MGBG induced an increase of this activity at 25°C and at 30°C, which diminished strongly up to 35°C. The fact that the inhibition of spermidine and spermine synthesis by CHA and/or MGBG provoked an induction of the enzyme of ethylene biosynthesis and a concomitant increase in ethylene production and a reversion of thermoinhibition in chick-pea seeds suggests that the ethylene and polyamine pathways compete during germination, placing polyamines in the possible role of an endogenous germination regulator possibly by modulating ethylene biosynthesis.

Content and Distribution of Free and Bound Polyamines in Embryonic Axes of Chick-Pea Seeds

Summary Changes in free (S) and bound polyamine contents were investigated in both the embryonic axes attached to the cotyledons and the embryonic axes excised from the whole chick-pea seeds before germination (isolated embryonic axes) during the first 24 hours at 25 °C, 30 °C or 35 °C. We also studied the distribution of these polyamines (PAs) along the embryonic axis of seeds germinated for 65 hours at 25 °C and 30 °C. The content in (S) polyamines was in general higher in embryonic axes separated from whole seeds than in isolated axes, suggesting a transport of these PAs from the cotyledons to the axis. At supraoptimal temperatures (S) polyamines decreased as germination proceeded, reaching higher levels than under the optimum temperature and the highest Spd and Spm accumulations were at 30 °C during the first 12 hours. The absence of cotyledons induced an important linkage of putrescine (Put) that strongly decrease with temperature. Methylglyoxalbis-(guanyl hydrazone) (MGBG) did not modify the total values of (S) and conjugated acid soluble (SH) polyamines, although it d change their distribution and increased conjugated acid insoluble (PH) polyamines.

Variations of the patterns of abscisic acid and proline during maturation of Nicotiana tabacum pollen grains

Summary Abscisic acid (ABA) and proline were evaluated during in situ development of anthers and pollen grains of Nicotiana tabacum . No conjugated forms of ABA were detected. The maximum accumulation of free ABA in the whole anther occurred at the mid-binucleate stage (MB), almost 95 % of the ABA being located in the pollen; this ABA distribution was not observed in the microspore (Mi) or mature (Ma) stages of the anther, in which the sporophytic tisue provided more ABA than did the pollen. A maximum in proline accumulation was also found at the MB stage of the anther; however, at the Ma pollen stage (when the ABA was scarcely detectable) the proline accumulation was maximum. The germination of the pollen grain was inhibited by exogenous ABA and stimulated by fluridone, a compound that inhibits carotenoid biosynthesis. The results of the present work suggest an important role for ABA in maturation and germination, as well as in the elongation of the pollen tube of N. tabacum .

Thermoinhibition alters the polyamine levels in cotyledons and embryonic axes during germination of stratified chick-pea seeds

Abstract The aim of the present work is to know how much the levels of both free and bound polyamines are altered in the cotyledons of Cicer arietinum by the presence or absence of embryonic axes during the first 24 h of germination under thermoinhibitory conditions (30°C). As an approach, embryonic axes and cotyledons were separated from dry seeds and also from germinated whole seeds. All classes of polyamines decreased both in cotyledons and embryonic axes from dry seeds during the stratification (0–4°C) period (12 month). At 25°C (control) all the polyamine types studied decreased in the cotyledons from germinated whole seeds, and accumulated in the isolated ones (embryonectomized). In cotyledons from germinated whole seeds, a supraoptimal temperature of 30°C provoked a rise in free spermine, free and bound spermidine, and putrescine bound to macromolecules, as compared to the control. However, all polyamines, except putrescine and spermine bound to small substances which remained unaffected, rose at 30°C in the embryonectomized cotyledons. In the embryonic axes separated from whole seeds, the free polyamine content was higher than in isolated ones, and vice versa for polyamines bound to low and high molecular compounds; this last effect being decreased by supraoptimal temperatures.

Modifications to polyamines provoked by short-chain saturated fatty acids in Cicer arietinum seeds

Short-chain saturated fatty acids (SCSFA), ranging from pentanoic (C5) to decanoic acid (C~0), when added at micromolar concentrations to the germination medium of chick-pea seeds, did not significantly affect radicle emergence, although fresh weight diminished. Only heptanoic acid (C7) and octanoic acid (Cs) stimulated ethylene production and free spermidine and spermine content, whereas putrescine reached a maximum with nonanoic acid (C9). All the SCSFA tested increased the polyamines (PAs) bound to substances of low Mr; however, only hexanoic acid (C6) and C9 acid induced a strong appearance of PAs bound to substances of high Mr. Proline synthesis was stimulated in the presence of C7 acid and C9 acid, but values lower than control were obtained with the other SCSFA studied. (C 1997 Elsevier Science Ltd. All rights reserved

Production of ethylene, its precursors and implied enzyme activities in isolated chickpea embryonic axes during the onset of growth

The embryonic axes of chickpea (Cicer arietinum) seeds were used to quantify 1-(malonyl)aminocyclopropane-1-carboxylic acid (MACC), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and some related enzymes during the initial 18 h imbibition period (anaerobic growth phase). Longer cold storage (stratification) of seeds produced higher levels of MACC and ACC. Maximum accumulation of MACC and malonyl-transferase activity occurred after 5 h of growth but MACC levels later became insignificant. ACC-synthase activity and endogenous ACC seem to reach a maximum 2 h after MACC accumulation. MACC-hydrolase activity was measured“in-vitro” and reached a maximum after 5.5 h of growth. These results suggest that endogenous MACC did not seem to be an end product; it may be involved in ACC production and the regulation of ethylene production before the emergence of the radicle. Ethylene-forming enzyme (EFE) activity reached a maximum after 12 h and ethylene production after 18 h of growth. The physiological implications of this temporal separation of MACC, ACC, ethylene and related enzymes is discussed.