Effect of Antioxidant BHT on the Proteolytic Apparatus of Aging Coleoptiles of Wheat Seedlings Grown in Light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in the light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

Apoptosis in Wheat Seedlings Grown under Normal Daylight

Apoptosis was observed in the coleoptile and initial leaf in 5-8-day-old wheat seedlings grown under normal daylight. Apoptosis is an obligatory event in early wheat plant ontogenesis, and it is characterized by cytoplasmic structural reorganization and fragmentation, in particular, with the appearance in vacuoles of specific vesicles containing intact organelles, chromatin condensation and margination in the nucleus, and internucleosomal fragmentation of nuclear DNA. The earliest signs of programmed cell death (PCD) were observed in the cytoplasm, but the elements of apoptotic degradation in the nucleus appeared later. Nuclear DNA fragmentation was detected after chromatin condensation and the appearance in vacuoles of specific vesicles containing mitochondria. Two PCD varieties were observed in the initial leaf of 5-day-old seedlings grown under normal daylight: a proper apoptosis and vacuolar collapse. On the contrary, PCD in coleoptiles under various growing (light) conditions and in the initial leaf of etiolated seedlings is only a classical plant apoptosis. Therefore, various tissue-specific and light-dependent PCD forms do exist in plants. Amounts of O2*- and H2O2 evolved by seedlings grown under normal daylight are less than that evolved by etiolated seedlings. The amount of H2O2 formed in the presence of sodium salicylate or azide by seedlings grown under normal daylight was increased. Contrary to etiolated seedlings, the antioxidant BHT (ionol) did not inhibit O2*- formation and apoptosis and it had no influence on ontogenesis in the seedlings grown under normal daylight. Thus, in plants grown under the normal light regime the powerful system controlling the balance between formation and inactivation of reactive oxygen species (ROS) does exist and it effectively functions. This system is responsible for maintenance of cell homeostasis, and it regulates the crucial ROS level controlling plant growth and development. In etiolated plants, this system seems to be absent, or it is much less effective.     

H1 Histone in Developing and Aging Coleoptiles of Etiolated Wheat Seedlings

It has been established that the DNA and H1 histone contents in aged coleoptile of 8-day-old etiolated wheat seedling are about 40 and 30%, respectively, lower than those in young seedlings. H1 histone in wheat seedlings is represented as six electrophoretically different subfractions. The ratios of H1 histone subfractions in wheat coleoptile and initial leaf are similar. In contrast to some animal cells, apoptosis in wheat coleoptile is not accompanied by changes in the set and ratios of H1 histone subfractions. Aging of coleoptiles is associated with a progressive diminution of the H1 histone and DNA contents. H1 histone/DNA ratio in aged coleoptile is 1.5-2-fold higher than that in the young organs. Therefore, the content of H1 histone in chromatin of coleoptile decreases with age more slowly than DNA content.     

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.     

Drought tolerance depends on the age of the spring wheat seedlings and differentiates patterns of proteinases

The majority of plant species lose their ability to tolerate severe water deficit after germination at the beginning of seedling growth, in the time of emergence of the radical from the seed. The experiment was designed to compare the differences in proteolytic response between 4-and 6-days old spring wheat (Triticum aestivum L.) seedlings of Eta cultivar, respectively tolerant and sensitive to severe drought inducing up to 90% water saturation deficit (WSD). In coleoptiles the changes of proteolytic activity had the same trend regardless on the seedlings age and increased about fourfold upon 85% WSD as compared to the control, from about 4 to 19 (U/mg protein h). The dehydration of roots of 4 day old seedlings resulted in sharp, fivefold activity increase at 85% WSD (from 11 to >50 U/mg protein h). In roots of 6 days old seedlings dehydrated to 55% WSD the proteolytic activity raised twofold and was about 2.5 times higher than in roots of 4 days old seedlings dehydrated to the same WSD. In coleoptiles of both the 4- and 6-days old seedlings subjected to drought appearance of new bands of serine proteinases was observed. Presented results indicate that roots are more sensitive to drought than coleoptiles, which brings an argument for breeders showing that programs involving roots phenotyping have a full biochemical rationale.     

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.     

The biophysical basis of cell elongation and organ maturation in coleoptiles of rye seedlings: implications for shoot development

The relationships between changes in irreversible and reversible organ length, turgor (P), osmotic pressure (pi), and metabolic activity of the cells were investigated in intact coleoptiles of rye seedlings ( Secale cereale L.) that were either grown in darkness or irradiated with continuous white light. Cessation of growth at day 4 after sowing was associated with an apparent mechanical stiffening of the cell walls. Turgor pressure was measured in epidermal and mesophyll cells with a miniaturized pressure probe. No gradient of turgor was found between the peripheral and internal cells. In juvenile (growing) coleoptiles, average turgor was 0.60 MPa and a negative water potential (P - pi) was established in these cells. Upon emergence of the primary leaf, turgor declined, but P was maintained at values of 0.43 and 0.52 MPa in 7-day-old light- and dark-grown coleoptiles, respectively. Water potential in non-growing cells approached zero. The rate of dark respiration and elongation growth were not correlated. Surgical removal of the mature coleoptile revealed that the erect position of the 7-day-old shoot was dependent on the presence of this sturdy, turgid organ sheath. It is concluded that, during the first week of seedling development, the pierced, metabolically active coleoptile fulfills an essential function as an elastic basal tube for the juvenile shoot.     

The Pool of Chlorophyllous Pigments in Barley Seedlings of Different Ages under Heat Shock and Water Deficit

The effects of a high temperature (3 h, 40°C) and water deficit (45 h on 3% PEG 6000) on the pool of chlorophyllous pigments in the leaves of 4-, 7-, and 11-day-old barley (Hordeum vulgare L.) seedlings were studied. Heating resulted in a decrease in the total content of chlorophylls (Chl) (a + b) in 4-day-old plants but not in the older leaves. Water deficit induced an increase in the pigment content in young seedlings but reduced it in the leaves of 11-day-old plants. In young seedlings, hyperthermia and dehydration affected similarly Chl (a + b) degradation, leading to a marked inhibition of the chlorophyllase (Chlase) activity hydrolyzing Chl to chlorophyllides and phytol. In old leaves, an activation of this enzyme was observed. The stress factors under study affected different stages of pigment biosynthesis. High temperature inhibited the activity of dark and light stages of Chl(a + b) biosynthesis. Dehydration did not change markedly the resynthesis of protochlorophyllide, while the enzymes of the light stage of Chl biosynthesis were activated in young but inhibited in old barley leaves. The results thus obtained allowed us to conclude that heat treatment and dehydration specifically affected the Chl biosynthesis. At the same time, the Chlase response was nonspecific.     

Ethylene and the Responses to Light of Rice Seedlings

The growth of rice seedlings (Oryza satira L.) in the presence of ethylene caused a change in the response to light of coleoptile elongation. In plants grown in air without added ethylene coleoptile elongation was promoted by red, far-red and yellow-green light only in very young seedlings; in older plants irradiation inhibited the growth of the coleoptile. The effect of growing plants in the presence of ethylene was to prolong the period during which light promoted coleoptile growth. Elongation of the first internode was inhibited by light whether or not the seedlings were grown in the presence of ethylene. A correlation existed between the growth effect of an irradiation and the initial decay rate of phytochrome which was established by the treatment. Regardless of wave length, light sources whose intensities were adjusted to produce a decay rate of about 10% per hour or less induced a moderate rate of coleoptile elongation which persisted for a relatively long period. Irradiation with red or yellow-green light of higher intensity which produced a higher rate of phytochrome decay induced a higher rate of coleoptile elongation, but growth stopped after several hours. Other observations, however, showed that one cannot establish a general simple correlation between the rate of elongation of rice coleoptiles under light and the status of measurable phytochrome in the plant.     

Synthesis of nucleosomal histone variants during wheat grain development

The synthesis and distribution of histone subfractions (variants) were investigated during early grain development and in mature tissues of wheat (Tritium aestivum L.). Histones were extracted from purified chromatin and separated by two-dimensional polyacrylamide gel electrophoresis. There were no detectable differences in the patterns of histone variants from immature grain (3–16 days after fertilization), from mature embryos, from coleoptiles and roots of 4-day-old, etiolated seedlings and from leaves of 10-day-old, light-grown seedlings. Wheat H2 histones are composed of families of closely related variants. H2A consists of three major variants, and H2B consists of two major and four minor variants. The synthesis of these variants during early grain formation was determined by calculating the specific activities of the [³H]lysinelabeled proteins synthesized between 3 and 10 days after fertilization. The rate of synthesis of the nucleosomal histones closely parallels the declining rate of cell division in developing grains. Our results indicate that all the recognized wheat histone variants are present in developing wheat grains from the earliest time investigated (3 days after fertilization) and persist with no detectable changes in relative quantities throughout grain development and in several mature tissues.     

Effect of the antioxidant ionol (BHT) on growth and development of etiolated wheat seedlings: control of apoptosis, cell division, organelle ultrastructure, and plastid differentiation

Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45·10^-5-2.27·10^-4 M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vac-uole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA ( = 1.718 g/cm³) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vac-uolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate dif-ferentiation of plastids and the Golgi apparatus in plants.     

Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo

Phytochrome A (phyA) mRNA abundance decreased rapidly in total RNA samples isolated from 4-day-old etiolated oat seedlings following a red light pulse. Putative in vivo phyA mRNA degradation products were detectable both before and after red light treatment. Cordycepin-treated coleoptiles were unable to accumulate the chlorophyll a/b-binding protein mRNA in response to red light, indicating that cordycepin effectively inhibited mRNA synthesis. In cordycepin-treated coleoptiles, phyA mRNA rapidly decreased in abundance, consistent with the hypothesis that phyA mRNA is inherently unstable, rather than being destabilized after red light treatment of etiolated oat seedlings.     

Apoptosis in Etiolated Wheat Seedlings: 2. Effects of the Antioxidant Butylated Hydroxytoluene and Peroxides

The antioxidant butylated hydroxytoluene (BHT, 50 mg/l, 2.27 × 10^–4 M) was found to prevent the development of characteristic signs of senescence and apoptosis in the cells of etiolated wheat (Triticum aestivum L.) seedlings. In particular, BHT blocked the apoptotic and age-induced formation of specific cytoplasmic mitochondria-containing vesicles in the coleoptiles. In contrast, the oxidants (H₂O₂ and cumene hydroxyperoxide) accelerated apoptosis (DNA fragmentation) in the coleoptiles and induced it in the first leaves, while in the control leaves, there were no signs of apoptosis. Thus, the programmed developmental apoptosis is controlled by the reactive oxygen species (ROS), and anti- and prooxidants can actively affect this process. In the coleoptile, BHT induced substantial changes in the ultrastructure of all cell organelles (nucleus, mitochondria, plastids, Golgi apparatus, and endoplasmic reticulum). It also induced the formation of unusual membrane structures in the cytoplasm and impaired nucleus and cell divisions. As a result, giant multilobed nuclei and multinuclear cells appeared. The effects of the antioxidant were tissue-specific: BHT did not noticeably affect cell ultrastructure in the first leaf. In roots of etiolated seedlings, BHT stimulated unusual plastid differentiation that resulted in the formation of chloroplasts, which is a phenomenon abnormal for roots. The BHT effects on the plant are evidently related to its antioxidant properties. Indeed, its structural analog, 3,5-di-tert-butyltoluene, which does not exhibit antioxidant properties, was physiologically inert. The BHT-controlled ROS evidently triggered apoptosis and produced age-dependent structural rearrangements of the cytoplasm and the formation of specific mitochondria-containing vesicles, which actively synthesize mtDNA. ROS inactivation by BHT is evidently responsible for BHT-induced changes in the structure of all cell organelles. Therefore, we believe that ROS control cell division (including nucleus division and cell-wall formation) and affect the differentiation of plastids and Golgi apparatus. In such a way, ROS effectively control plant growth and development.     

Ultrastructural study of an olive necrotic mutant of maize

Summary Ultrastructural surveys, carried out into an olive necrotic maize mutant (Neuffer E 283 B) grown under a 16 hours photoperiod, have shown remarkable morphological alterations in the plastids. Such alterations, affecting both the mesophyll and the bundle sheath plastids, appear to be photodependent. Quite normal etioplasts are present in dark-grown mutant seedlings. Moreover, light appears also to inhibit the overall growth of mutant plants. 12-day-old mutant plants grown under illumination are 4 cm in comparison with the 10 cm of the wild type, while corresponding dark-grown seedlings of both types are 12 cm high.Supported by a grant of C.N.R.     

Establishment of far‐red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene

A transgenic wheat line over‐expressing an oat phytochrome A gene under the control of the constitutive maize ubiquitin promoter was generated using a biolistic particle delivery system from immature wheat embryos. The resulting line showed increased levels of total phytochrome A protein in both dark‐grown and light‐grown plants. When grown under continuous far‐red light, seedlings of this line showed additional inhibition of the coleoptile extension in comparison with wild‐type seedlings. Unlike the response of wild‐type seedlings to continuous far‐red, this additional inhibition was dependent on fluence rate and was not observed under half‐hourly pulses of far‐red delivering the same total fluence as the continuous irradiation treatment. These observations suggest that increase in phytochrome A levels in wheat leads to the establishment of a far‐red high irradiation reaction in this monocotyledonous plant. Exposure to continuous red light caused a similar inhibition of coleoptile extension in both the wild types and the transgenic seedlings. When wild‐type seedlings were grown under continuous far‐red, their coleoptiles remained completely colourless and first leaves remained tightly rolled. In contrast, transgenic seedlings grown in the same conditions produced significant levels of anthocyanins in their coleoptiles and their first leaves became unrolled. Taken together, our data suggest that the increased levels of phytochrome A in wheat can change the type of response of some developmental processes to light signals, leading to the generation of a high irradiance reaction which is otherwise absent in the wild types under the conditions used.     

Characterization of protein and transcript levels of the chaperonin containing tailless complex protein-1 and tubulin during light-regulated growth of oat seedlings

In grass seedlings the network of cortical microtubules is reorganized during light-dependent growth of coleoptiles and mesocotyls. We investigated the effects of light-dependent growth on the relative steady-state levels of the mRNAs and protein levels of alpha-tubulin and the epsilon-subunit of the chaperonin containing tailless complex protein-1 in oat (Avena sativa) coleoptiles, which were grown in different light conditions to establish different growth responses. The soluble pools of the epsilon-subunit of the chaperonin containing tailless complex protein-1 and alpha-tubulin decreased in nonelongating coleoptiles, suggesting that the dynamics of the light-regulated soluble pool reflect the processes occurring during reorganization of cortical microtubules. The shifts in pool sizes are discussed in relation to the machinery that controls the dynamic structure of cortical microtubules in plant cells.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)