Isa Grilli

Hydrolysis of Gliadin during Germination of Wheat Seeds

Summary The in vivo degradation of gliadin has been studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. Both analyses show the appearance of a small number of degradation products and the early disappearance of some gliadin components during the course of germination. These components thus appear to be subject to preferential breakdown, the w-gliadin fraction being the first to be degraded. The in vitro degradation of total gliadin or selected gliadin fractions, performed by the main endopeptidase appearing during wheat germination, reveals a transient accumulation of intermediates, which were subsequently degraded to small peptides. These data suggest that the relatively small amount of intermediates formed during wheat germination might be due to the concerted action of the cysteine proteinase and of the carboxypeptidases. Since these enzymes have high activities during the course of seed germination, they might cause a rapid degradation of intermediates before they can accumulate.

Rate of synthesis of spermine and spermidine in germinating seeds of Glycine, Helianthus and Triticum.

The spermine, spermidine, and the total protein content of embryos or embryonic axes from Triticum durum, Helianthus annuus, and Glycine max seeds at different times of early germination was evaluated. Mitotic activity of root-tip meristems from germinating seeds was also determined. The hypothesis is suggested that differences in polyamine and protein pattern during early germination could be correlated with the onset of mitotic activity and with the different characteristics of the seeds assayed.

Responses to desiccation injury in developing wheat embryos from naturally- and artificially-dried grains.

Grains of wheat (Triticum durum Desf. cv. Cappelli) were collected at different stages of maturation. To test whether the response to desiccation injury is correlated with physiological stage and/or water content, one lot of grains at each stage was artificially dried until its water content was comparable to that of the subsequent stage. Several stress parameters were analysed in embryos isolated from naturally-and artificially-dried grains. In particular, the content of dehydrins, hydrogen peroxide and the activity of ribonucleases and of antioxidant enzyme were studied. Neutral ribonucleases, unlike acidic ones, seemed to be correlated with grain water content. In parallel with these analyses the presence of dehydrins was also examined. Dehydrin accumulation was found to vary with drying rate, and more isoforms of dehydrin were present in artificially dried than in naturally-dried grains. Artificial drying resulted in accumulation of hydrogen peroxide, while the content of this molecule was much lower in embryos from naturally fully-ripened grains. While the activities of ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), glutathione reductase (GR, EC 1.6.4.2), and glutathione peroxidase (GPX, EC 1.11.1.9), were generally higher early in maturation, the activity of catalase (CAT, EC 1.11.1.6) increased in mid-maturation and decreased in fully-ripe embryos. Different protective mechanisms seem to act in combination but their relative importance changes as maturation progresses and in response to the drying method.

Metabolism in Seed Ripening: Protein and poly(A)+ RNA Pattern in developing embryos of Triticum durum

Abstract Pattern of proteins and poly(A) + RNA synthesis in Triticum durum embryos during their development and in after-ripened seeds was studied. The aim was to evaluate differences as to the polyadenylated RNA in relation to the ripening and dormancy of caryopses. Developing embryos show a progressive increase of protein and poly(A) + contents while, during the after ripening period, a decrease of both occurs. With these events a syncronous incorporation pattern of labeled precursors does not coincide: therefore it is possible that, during embryo development, a progressive utilization of both proteins and poly(A) + RNA occurs. Moreover, the poly(A) + RNA of dormant dry embryos exhibits a template capability higher than of the after-ripe ones. On the contrary, when the after ripe embryos are from imbibed seeds, their transla-tional capability is higher. Some hypotheses about the different messengers for proteins probably involved in the maintaining of seed dormancy are discussed.

Metabolism in Wheat Seed Dormancy: Polyamine Content during Early Germination

Summary Spermine and spermidine content of embryos from dormant and non-dormant wheat seeds, during the early phases of germination was analyzed. Treatment of dormant seeds with GA3 and of non-dormant seeds with GA3 and ABA was also performed to verify, at a metabolic level, the consequences of the stimulating effects of GA3 on dormant and the inhibitory activity of ABA on non-dormant seeds. Polyamine content of treated and control seeds in relation to protein content, physiological state of seeds and treatment with plant growth regulators are discussed.

Ageing in embryos from wheat grains stored at different temperatures: oxidative stress and antioxidant response

In the present work we studied oxidative stress as an important cause of seed deterioration during ageing in embryos from durum wheat grains stored at room temperature and at low temperature (10°C). The protective role of low temperature on seed viability was confirmed. The increase of hydrogen peroxide content during dry storage was strongly correlated with the decrease of germinability. Ascorbate and glutathione showed a good correlation with grain germinability and significantly increased upon imbibition, in particular in embryos from viable grains. Ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX) and catalase (CAT) were studied quantitatively (enzymatic assays). APX, GR, and GPX were also studied qualitatively by native PAGE. The enzymes were active in dry, still viable, embryos whereas no activity was detected in non-viable embryos. With the exception of APX, all enzymatic activities decreased upon imbibition. The study of grains stored in different conditions indicated a negative correlation between the efficiency of the antioxidant enzymatic machinery and the age of the grain. The differences detected in differently stored materials confirmed that both germination parameters and the length of storage period are important in determining grain condition.

Dormancy and germination in wheat embryos: ribonucleases and hormonal control

Acidic and neutral ribonucleases (RNases) were studied in embryos of Triticum durum cv. Cappelli and the effects of abscisic acid (ABA) and gibberellic acid (GA3) were analysed. RNases activities increased during germination and were comparable in dormant and non-dormant embryos imbibed for 24 h. ABA generally inhibited ribonucleolytic activities, while GA3 only affected dormant embryos. To assess whether changes in RNase activities during germination or following hormonal treatment required new transcriptional or translational action, cycloheximide or cordycepin were used. The action of inhibitors of acidic RNase activity was found only in non-dormant-embryos. Findings obtained in the present work concur with a change of the ribonucleolytic pattern in the shift from dormant to non dormant metabolism.

Polyamine accumulation in aged wheat seeds

The present work was conducted to evaluate the content of the main polyamines (Spm, Spd, Put) in a series of naturally aged durum-wheat seeds as well as the activities of the enzymes ODC and ADC involved in their biosynthesis. In dry seeds the content of polyamines, especially that of Spd, rose during ageing till 6 years and then declined sharply. However, an increase of PA content upon imbibition was observed only with the youngest seeds, while a decrease was found in the older ones.The activities of ODC and ADC differed in aged seeds, the ODC activity being constant and lower than the ADC in the course of seed ageing. The ADC increased till the early ageing and decreased then in the very old, ungerminating seeds. Imbibition increased both enzyme activities in the youngest seeds only, in the older ones rather a decrease and changed ADC/ODC ration was found.The obtained results are discussed in relation to the participation of these enzymes in the biosynthesis of polyamines during seed ageing and in the course of plant senescence or stress.

Effects of GA3 treatment on the synthesis of poly(A)-rich RNA in wheat dormant embryos

Summary Gibberellic acid (GA 3 ) is capable to improve seed germination and seedling growth of dormant (D) wheat seeds. Total RNA content and synthesis rate of D embryos, lower than in non-dormant ones (ND), do not increase when GA 3 is applied during early 10 h of seed soaking. Synthesis of poly(A)rich RNA is stimulated after 8 h of hormonal treatment, but the response to GA, is similar for both D and ND embryos. Dry D embryos exhibit a content of poly(A)-rich RNA higher than ND embryos, and differences in the rate of synthesis exist during early germination. This pattern and the effects of GA 3 treatment in relation to different physiological state of seeds are discussed.

Ribonucleases during ripening and after-ripening in Triticum durum embryos

Summary Total and polyadenylated RNA content changes during ripening and after-ripening in embryos of Triticum durum cv. Cappelli. In this work, RNase activity was studied by means of enzymatic assays and activity staining SDS-PAGE in embryos at different stages of maturation and after-ripening. Qualitative and quantitative changes of RNases were found. Of particular interest was the continuous presence of acidic RNase in embryos, and the disappearance of neutral RNase activities during after ripening. Salt-inhibited RNases disappeared 42 days after anthesis, while salt-stimulated RNases with higher mobility disappeared in fully ripened embryos. Salt-stimulated RNases with lower mobility disappeared during after-ripening. The disappearance of neutral RNase activities during ripening may be related to the different physiological status induced by dehydration.