Luciano Galleschi

Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant

A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, alpha- and gamma-tocopherol amount and antioxidant capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46 degrees C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing. Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals, mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but the gamma-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology can be assigned to phytic acid, that is, protection against oxidative stress during the seeds life span. As in maize kernels the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular relevance in this crop.

Major proteinase hydrolysing gliadin during wheat germination

Abstract A proteinase, representing the bulk of the enzyme activity for the hydrolysis of gliadin, was extracted from endosperms isolated from germinated seeds (four days) and was purified by ion-exchange chromatography and preparative isoelectric focusing. The optimal pH for gliadin hydrolysis was 4.25. The M r , determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 30 000; the isoelectric point was 4.5. The enzyme activity was totally inhibited by E-64 and cystatin, while inhibitors of other classes of proteinases were barely effective or ineffective. The activity was stimulated by sulphhydryl compounds. The proteinase hydrolysed to small peptides the gliadins from durum and soft wheat seeds. Other protein substrates were weakly degraded or not degraded. The proteinase appears to belong to the cysteine class and to play a key role in the initial mobilization of the main reserve protein in the starchy endosperm.

Lipid evolution during development and ripening of peach fruits

Changes in the phospholipid, glycolipid, diacyl and triacylglycerol, free fatty acid and free sterol classes were examined during the development and ripening of Prunus persica cv. Redhaven peach fruit. Total lipid and lipid class contents decreased until 100 days after full bloom (AFB). While phospholipids and glycolipids decreased in the following stages, diacyl- and triacylglycerols increased. The analysis of each lipid class showed that the predominant fatty acids were 16:0 and 18:2. The double bond index showed, in each class, a general increase during maturation. The free sterol content continuously declined during maturation with the exception of samples at 100 days AFB, which showed an increase in the free sterol to phospholipid molar ratio. The main changes observed in lipids occurred mostly between the climacteric and postclimacteric stages.

Effect of Salinity on Water Relations, Sodium Accumulation, Chlorophyll Content and Proteolytic Enzymes in a Wild Wheat

The effect of 50 to 200 mM NaCl on two lines (CP with solid stem and CV with hollow stem) of ×Haynaldoticum sardoum was studied. NaCl significantly reduced root and shoot fresh and dry masses, root length and less markedly shoot length of CP and CV plants. The sodium accumulated in the leaves in relation to the concentration of NaCl and length of the treatment; CP leaves contained twice as much sodium as CV leaves. The leaf chlorophyll a/b ratio was not affected by NaCl. NaCl decreased the leaf water and osmotic potentials. The pressure potential increased due to the increased concentration of dissolved solutes in the leaf, particularly sodium. The proteinase and exopeptidase activities increased during NaCl treatment.

Purification, characterization and activation by anions of an aspartic proteinase isolated from bran of soft wheat

Abstract An aspartic proteinase, contained in wheat bran, was purified to homogeneity by ion-exchange, gel permeation, affinity chromatography and electro-endosmotic preparative electrophoresis. The optimal pH for the activity of the purified enzyme was 3.3, as measured by the hydrolysis of a specific chromophoric substrate, and the Km value was 0.375 mM. The apparent molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 66.5 kDa. The enzyme activity was completely inhibited by pepstain, whereas other proteinase inhibitors were ineffective. The enzyme hydrolyzed at pH 3.3 endogenous globulin, releasing fragments with molecular weights over 20 kDa. The purified proteinase was activated by anions, particularly sulfate, and this effect was instantaneous, operative when the reaction was initiated, and insensitive to changes in the ionic strength of the reaction medium. A comparison with other aspartic proteinases of animal origin revealed that the enzyme modulation by anions was a characteristic of the plant enzyme alone. Finally, circular dichroism analyses showed a conformational change in the plant enzyme in the presence of sulfate. These data suggest a new control mechanism for modulating proteolytic activity in seeds, and the participation of aspartic proteinase in initiating the degradation of globulin stored in the aleurone layer.

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.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

Influence of Age and Storage Temperature on RNA Metabolism in Durum Wheat Seeds

Summary Pattern and metabolic activity of embryo RNA from wheat seeds of different ages (2, 6, 9 year-old seeds) with a different germination capacity in relation to storage conditions has been studied. The embryos from 9 year-old seeds stored at laboratory conditions show a degraded RNA which is also incapable of incorporating ( 3 H)-adenosine. Storage at 10°C permits the 9 year-old seeds to maintain embryo RNA classes which are similar to those of younger seeds, and a poly(A) + rich RNA fraction with a high ( 3 H)-adenosine incorporation. The 6 year-old embryos from seeds stored at laboratory conditions have a high germination capacity but low germination energy. They show a degraded RNA, with a prevalence of a light class of RNA, and an ( 3 H)-adenosine incorporation which is lower than in the case of the controls (2 year-old seeds). Storage at 10°C shows that the RNA of the 6 year-old embryos maintains characteristics and an incorporation capacity which are similar to unaged seeds. The experiments also show the protective action of low temperature against degradation processes during storage and ageing. The paper also discusses the relationships between the structural and functional stability of RNA and seed germination and seedling growth.

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The Km for SSA and for NAD are 7.4x10-6 M and 2x10-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.