Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley

Chlorophyf l-free preparations of plasma membranes from leaves of barley (Hordeum vulgare L. cv. Kristina) and spinach (Spinada oleracea L. cv. Viking II) were obtained by partition in an aqueous dextran-polyethylene glycol two-phase system. CJlu-can synthetase II (EC 2.4,1.34), a marker for the plasma membrane, was highly enriched in both preparations. Silicotungstic acid, a specific stain for the plasma membrane, indicated a purity close to 100% for the barley preparation. Both plasma membrane preparations contained a light-reducible b-cytochrome, as shown by low temperature spectroscopy. The plasma membranes had a tow protein content compared to the bulk of intracellular membranes. The polypeptide composition of the barley and spinach plasma membranes showed striking similarities, with.the most prominent polypeptides in the 49-58 kdalton region, and some further prominent bands in the 30 kcialton region. Some high molecular weight polypeptides in the 73-110 kdalton region were also typical for the plasma membranes compared to the microsomal fractions.     

Ultrastructural changes in aging leaves of a light-grown achlorophyllous mutant of barley

The pattern and sequence of cellular degradation during the course of leaf senescence remains obscure and the nature of the trigger that induces cell senescence is unknown. In order to probe the pre-mortem phase of senescence temporal changes in cell ultrastucture were studied in aging leaves of light-grown achlorophyllous Hordeum vulgare L. cv. Dyan mutant seedlings. Electron microscope examination of the ultrastructure of mesophyll cell plastids revealed the absence of ribosomes and a highly disorganized prolamellar body. Both the number and size of plastoglobuli increased with aging and this change coincided with depletion of starch grains and dilation of lamellar membranes. Aging of mesophyll cells occurred coincident with a decline in ribosome content of the cytoplasm and loss of matrix granularity. Loss of ribosomes associated with the outer nuclear envelope membrane and a reduction in chromatin were also apparent. Only after 10 days was there evidence of loss of internal membrane integrity and swelling of mitochondrial cristae. Compartmentation was thus maintained during the aging process with membrane dissolution occurring late in senescence. These results suggest that an inability to produce chlorophyll and carotenoids and form thylakoid stacks due to the absence of plastid ribosomes, contributes to the rapid onset of senescence in light-grown achlorophyllous seedlings. Furthermore, disruption of chloroplast ribosome synthesis/assembly may constitute part of the plastid signal involved in triggering cell senescence.     

Protein synthesis by chloroplasts during the senescence of barley leaves

Chloroplasts were isolated from senescent leaf segments of barley ( Hordeum vulgare L. var. Mozoncillo) and assayed for protein synthesis. Protein synthesis activity of the chloroplasts greatly increased after 10–20 h of incubation of leaf segments in the dark in spite of an intense degradation of chloroplast rRNA. The rise in the activity of protein synthesis was more pronounced when kinetin was present in the incubation medium. However, as deduced from SDS-polyacrylamide gel electrophoresis of the products, different proteins were synthesized under the two conditions of incubation of the leaf segments. The activity of protein synthesis of the chloroplasts decreased during the first hours of incubation of the leaf segments in the light.
Cutting and incubation in the dark of the leaf segments enhanced the synthesis of a few proteins also formed by chloroplasts in attached senescing leaves. Hormone and senescence treatments changed the type and the rate of the protein synthesized by chloroplasts, which suggests that hormones may control senescence through a modulation of the protein synthesized by the chloroplasts.     

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - CP A/B the major light-harvesting complex associated with photosystem II - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenyl carbazide - MV methyl viologen - PAR photosynthetically active radiation - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TEMED N,N,N,N-tetramethylethylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 9949 of the Journal Series of the North Carolina Agricultural Research Service, Raleight, NC 27695-7601.     

Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Uptake of amino acids and peptides by developing barley embryos

Developing embryos of barley ( Hordeum vulgare L. cv. Bomi) detached 21–27 days after anthesis took up 1 mM [¹⁴C]-glutamine at pH 5 and 30°C at a rate of about 20 nmol embryo^−l h⁻¹ (5 μol g⁻¹h⁻¹). The uptake was inhibited by about 50% by di-nitrophenol and by about 80% by 300 m M unlabelled glutamine or alanine. The bulk of the uptake appeared, therefore, to be due to carrier-mediated active transport. The pH optimum of the uptake was 4.5. Leucine, proline, lysine, arginine and as-paragine were taken up at approximately similar rates as glutamine, and they also inhibited the uptake of glutamine. This, suggests that the uptake of glutamine was at least partly due to an unspecific carrier(s) also shared by other amino acids. The embryos also took up the dipepti.de glycykarcosine; the rate was about 6 nmol embryo⁻¹h⁻¹ (1.5 μol g⁻¹h⁻¹) (2 mM glycylsarcosine, pH 4.5, 30°C). The uptake was inhibited by about 70% by dinitrophenol or by 300 m M glycylglycine. This indicates that the bulk of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was about 4.5.
The rates of glutamine and glycylsarcosine uptake increased during the early and middle stages of embryo development (until day 28 after anthesis), but decreased towards the end of the maturation of the grain. These changes, as well as the relatively high activities, suggest that carrier-mediated active uptake of amino acids, and possibly also that of peptides, plays a role in the nutrition of the developing embryo.     

Polar lipid composition of chloroplast thylakoids isolated from leaves grown under different lighting conditions

The polar acyl lipid composition was determined for samples of chloroplast thylakoids isolated from Pisum sativum plants grown at light intensities of 50 and 300 E·m^-2·s^-1 and from Aesculus hippocastanum leaves taken from shade or sun environments. Lighting conditions had no major effect on lipid class composition except for a small increase in the amount of monogalactosyldiacylglycerol relative to other lipids in low compared with high light and shade compared with sun conditions. The thylakoids from low light and shade environments also had, relative to those from high light and sun conditions, a substantial decrease in the level of trans-hexadecenoic acid in phosphatidyglycerol. In parallel with this there were lower lipid to chlorophyll ratios, higher overall fatty acid unsaturation, lower chlorophyll a to b ratios and increased relative levels of light harvesting chlorophyll a/b polypeptides as expected for an increase in the degree of thylakoid appression. With this in mind, our results on lipid class composition and content of trans-hexadecenoic acid are discussed in the context of the lateral distribution of lipids within the plane of membrane.Abbreviations DGDG digalactosyldiacylglycerol - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b - MGDG monogalactosyldiacylglycerol - MPL minor phospholipids - PS1 photosystem one - PS2 photosystem two - SDS sodium dodecyl sulphate - SL sulphoquinovosyldiacylglycerol     

Diurnal variations of nitrate reductase activity and stability in barley leaves

Diurnal variations of nitrate reductase (NR) activity and stability have been studied in leaves of barley seedlings ( Hordeum vulgare L. cv. Herta) grown in an 8 h light/16 h darkness regime. Stability (decay) of NR was tested both in the extracts and in the plants. In the morning, when the plants were transferred to light, NR activity increased rapidly during the first hour and then remained constant. After the photoperiod, activity decreased rapidly during the first hour of darkness and then remained fairly constant during the rest of the dark period. The high NR activity during the photoperiod was associated with low NR stability both in the extracts and in the plants. On the other hand the low NR activity during the dark period was associated with high stability in the extracts and in the plants.     

Regulation of nitrate and nitrite reduction in barley leaves

Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O₂ and N₂) or anaerobic (N₂) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N₂ or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed.     

Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Isolation, characterization and expression of a cDNA coding for a jasmonate-inducible protein of 37 kDa in barley leaves

In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate-inducible proteins (JIPs). In the present study, a new jasmonate-inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate-treated barley leaf segments. The open reading frame (ORF) encodes a 39-9 kDa polypeptide which cross-reacts with antibodies raised against the in vivo JIP-37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C-terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP-37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP-23. The expression pattern of the JIP-37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α-linolenic acid and 12-oxophytodienoic acid, we hypothesize that there is a stress-induced lipid-based signalling pathway in which an endogenous rise of jasmonate switches on JIP-37 gene expression. Using immunocytochemical techniques, JIP-37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.     

Diurnal variations of nitrite reductase, glutamine synthetase, glutamate synthase, alanine aminotransferase and aspartate aminotransferase in barley leaves

Diurnal variations of in vitro activity of 5 enzymes of nitrogen metabolism were studied. Barley ( Hordeum vulgare L. cv. Herta) seedlings were grown in 8 h short days, in daylight or under fluorescent lamps. During, the photoperiod nitrite reductase (EC 1.7.7.1) increased by an average of 18% in daylight and 10% under fluorescent lamps. Glutamine synthetase (EC 6.3.1.2) activity increased by 14 and 10%, respectively. The increase in enzyme activity reflected the overall increase in soluble proteins which was 8% in daylight and 3% under fluorescent lamps. Alanine aminotransferase (EC 2.6.1.2) increased by 82% in daylight and 37% under fluorescent lamps. Desalting of the extracts did not alter the enzyme activity and thus supported the assumption that changes in extractable enzyme activity are due to changes in the amount of (active) enzyme protein. Glutamate synthase (EC 1.4.7.1) activity did not show regular diurnal variations, and aspartate aminotransferase (EC 2.6.1.1) activity was almost constant.     

Effect of low above-zero temperature on the content of low-molecular antioxidants and activities of antioxidant enzymes in green barley leaves

We studied the effect of low above-zero temperature (2°C) on the content of low-molecular antioxidants (ascorbic acid, glutathione, and carotenoids) and also activities of antioxidant enzymes (ascorbate peroxidase, APO; catalase, CAT; glutathione reductase, GR; and superoxide dismutase, SOD) in green barley (Hordeum vulgare L.) seedlings. Under stress conditions, the content of low-molecular antioxidants, especially that of reduced ascorbate form, increased. Low-temperature stress activated APO, CAT, GR, and SOD. First enzymes responding to the action of stress factor were APO and CAT, i.e., enzymes neutralizing hydrogen peroxide in plant cells, which indicated H₂O₂ active generation at low temperature. Cytoplasmic SOD was more active than its chloroplast isoforms. This indicates that oxidative process initiation under low-temperature stress occurred more active in the cytosol. After termination of stress-factor action, the content of total ascorbate, glutathione, and carotenoids reduced rapidly to the level close to the initial one. During post-stress period, the amount of reduced ascorbate declined as well; however, it remained at the level higher than the initial one. Activities of APO and CAT dropped sharply; activities of GR and SOD reduced gradually. Thus, reduced ascorbate, APO, and CAT play an important role in plant cell defense against above-zero temperatures close to zero; reduced ascorbate, GR, and SOD are especially important during post-stress period.     

Changes of chloroplasts density and heterogeneity during the senescence of barley leaves

The equilibrium density of chloroplasts from barley (Hordeum vulgare L. cv. Hassan) was analyzed by sucrose gradient centrifugation. Natural and detachment-induced leaf senescence were associated with a decrease in density and an increase in heterogeneity of the chloroplast population. Treatments (with growth regulators and light) which retarded or accelerated senescence, respectively, retarded or accelerated chloroplast density decrease. Accelerators as well as retardants of senescence decreased the heterogeneity of the chloroplast population.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants

A study was performed to examine whether or not betaine (glycinebetaine), a compatible solute, is accumulated in response to cold stress and is involved in mechanisms that protect plants from freezing injury. For this purpose, we used near-isogenic lines of barley, with each line differing only in a single gene for the spring type of growth habit; the various lines were produced by back-crosses to a recurrent cultivar of the winter type. The winter type of growth habit requires a low temperature for triggering of flower development (vernalization), whereas the spring type does not. Betaine was accumulated to five times the basal level over the course of 3 weeks at low temperature (5 °C) in the winter-type cultivar and in a spring-sh line having the sh gene for the spring-type growth habit, but the level was only doubled in the spring-Sh3 line, which carried the Sh₃ gene for the spring-type growth habit. Among near-isogenic lines of the same cultivar, the levels of betaine accumulated in leaves at low temperature were well correlated with the percentages (on a dry weight basis) of green leaves that survived freezing injury (-5 °C). This observation indicates the possibility, separate from the recognized role of betaine in the response to salinity and/or drought, that betaine accumulates in response to cold stress and that the accumulation of betaine during cold acclimation is associated to some extent with freezing tolerance in leaves of barley plants.     

Involvement of the phosphoinositide signaling pathway in the anti-senescence effect of low-concentration stressors on detached barley leaves

The effect of low concentration of some stress-inducing compounds of different toxicity and chemical nature like Pb and Ti salts or DCMU on the senescence of chloroplasts was investigated in detached primary leaves of barley (Hordeum vulgare cv. Omega). These agents stimulated chlorophyll accumulation, photosynthetic activity ((14)CO (2) fixation), and decreased the number of plastoglobuli in chloroplasts compared to the control, thus delaying senescence. Low-concentration stressors did not increase the level of active cytokinins of leaves during the treatment. Lithium and stearoylcarnitine chloride inhibited the stimulating effect of stressors. This points to the involvement of the PIP (2)-IP (3)/DAG signal transduction pathway in generation of the specific responses.