Primary Leaf Of Wheat Research Articles

The physiological importance of photosynthetic ferredoxin NADP+ oxidoreductase (FNR) isoforms in wheat

Ferredoxin NADP+ oxidoreductase (FNR) enzymes catalyse electron transfer between ferredoxin and NADPH. In plants, a photosynthetic FNR (pFNR) transfers electrons from reduced ferredoxin to NADPH for the final step of linear electron flow, providing reductant for carbon fixation. pFNR is also thought to play important roles in two different mechanisms of cyclic electron flow around photosystem I; and photosynthetic reductant is itself partitioned between competing linear, cyclic, and alternative electron flow pathways. Four pFNR protein isoforms in wheat that display distinct reaction kinetics with leaf-type ferredoxin have previously been identified. It has been suggested that these isoforms may be crucial to the regulation of reductant partition between carbon fixation and other metabolic pathways. Here the 12 cm primary wheat leaf has been used to show that the alternative N-terminal pFNRI and pFNRII protein isoforms have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress. More specifically, the results obtained suggest that the alternative N-terminal forms of pFNRI have distinct roles in the partitioning of photosynthetic reductant. The role of alternative N-terminal processing of pFNRI is also discussed in terms of its importance for thylakoid targeting. The results suggest that the four pFNR protein isoforms are each present in the chloroplast in phosphorylated and non-phosphorylated states. pFNR isoforms vary in putative phosphorylation responses to physiological parameters, but the physiological significance requires further investigation.

Expression of multiple forms of ferredoxin NADP+ oxidoreductase in wheat leaves

In higher plants there are two forms of ferredoxin NADP(+) oxidoreductase (FNR), a photosynthetic pFNR primarily required for the photoreduction of NADP(+), and a heterotrophic hFNR which generates reduced ferredoxin by utilizing electrons from NADPH produced during carbohydrate oxidation. The aim of this study was to investigate the presence of multiple forms of FNR in wheat leaves and the capacity of FNR isoforms to respond to changes in reductant demand through varied expression and N-terminal processing. Two forms of pFNR mRNA (pFNRI and pFNRII) were expressed in a similar pattern along the 12 cm developing primary wheat leaf, with the highest levels observed in plants grown continuously in the dark in the presence (pFNRI) or absence (pFNRII) of nitrate respectively. pFNR protein increased from the leaf base to tip. hFNR mRNA and protein was in the basal part of the leaf in plants grown in the presence of nitrate. FNR activity in plants grown in a light/dark cycle without nitrate was mainly due to pFNR, whilst hFNR contributed significantly in nitrate-fed plants. The potential role of distinct forms of FNR in meeting the changing metabolic capacity and reductant demands along the linear gradient of developing cells of the leaf are discussed. Furthermore, evidence for alternative N-terminal cleavage sites of pFNR acting as a means of discriminating between ferredoxins and the implications of this in providing a more effective flow of electrons through a particular pathway in vivo is considered.

Ultraviolet‐B radiation reduces the rates of cell division and elongation in the primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman)

AbstractThe primary leaf of wheat (Triticum aestivum L. cv Maris Huntsman) was used as a model system to examine how elevated ultraviolet‐B (UV‐B; λ= 280–320 nm) radiation affected growth. A reduction in the rate and duration of growth of the primary leaf, in response to UV‐B, was the result of changes in both the rate and extent of cell division and elongation. UV‐B reduced the proportion of mitotically active cells (mitotic index) and increased the time taken for cell division (cell doubling time). Thus the supply of cells into the elongation zone was reduced, and this, coupled to a reduction in the rate of elongation, resulted in reduced leaf growth. This analysis of the spatial distribution of growth provided a means of calculating the age of cells within the leaves. Cells of UV‐B‐treated leaves were found to age more quickly than those of the controls. This analysis will enable future studies to take account of age‐related changes when interpreting the response of plants to any number of environmental stresses that affect leaf development.

The Distinctive Pattern of Photosystem 2 Activity, Photosynthetic Pigment Accumulation, and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content of Chloroplasts along the Axis of Primary Wheat Leaf Lamina

Wheat (Triticum aestivum L. cv. Sonalika) seedlings were grown in Hoagland solution. Primary leaves were harvested at 8, 12, and 15 d and cut into five equal segments. Contents of photosynthetic pigments and proteins, and photosystem 2 (PS2) activity increased from base to apex of these leaves. Chlorophyll (Chl) content was maximum at 12 d in all the leaf segments, but PS2 activity showed a gradual decline from 8 to 15 d in all leaf segments. In sharp contrast, the CO2 fixation ability of chloroplasts increased from 8 to 15 d. CO2 fixation ability of chloroplasts started to decline from base to apex of 15-d-old seedlings, where the content of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBPCO-LSU) increased acropetally. RuBPCO-LSU content was maximum in all the leaf segments in 12-d-old seedlings. This shows a distinctive pattern of PS2, Chl, CO2 fixation ability of chloroplasts, and RuBPCO-LSU content along the axis of leaf lamina during development and senescence. RuBPCO-LSU (54 kDa) degraded to fragments of 45, 42, 37, 19, and 16 kDa products which accumulated along the leaf axis during ageing of chloroplasts. Thus the CO2 fixation ability of chloroplasts declines earlier than PS2 activity and photosynthetic pigment contents along the leaf lamina.

Senescence Retarding Effect of Metal Ions: Pigment and Protein Contents and Photochemical Activities of Detached Primary Leaves of Wheat

Al3+ significantly delayed the loss of chlorophyll (Chl), protein, and carotenoids when compared to K+ and Mg2+ during dark-induced senescence of detached primary leaves of Triticum aestivum. Thylakoid membranes isolated from Al3+ - treated leaves showed a better retention of photosystem (PS) 2, PS1, and whole chain electron transport activities than thylakoids of K+- or Mg2+-treated leaves. These ions protected the electron transport activities and restored the DCMU-dependent fluorescence increase of thylakoid membranes in a valency-dependent manner. Al3+ also delayed the change of excitation energy distribution during senescence.

The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is fragmented into 37-kDa and 16-kDa polypeptides by active oxygen in the lysates of chloroplasts from primary leaves of wheat.

Lysates of chloroplasts isolated from wheat (Triticum aestivum L. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to 60 min in light (15 mumol quanta m-2 s-1), and degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) was analyzed by applying immunoblotting with site-specific antibodies against the N-terminal, internal, and C-terminal amino acid sequences of the LSU of wheat Rubisco. The most dominant product of the breakdown of the LSU and that which was first to appear was an apparent molecular mass of 37-kDa fragment containing the N-terminal region of the LSU. A 16-kDa fragment containing the C-terminal region of the LSU was concomitantly seen. This fragmentation of the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline. The addition of active oxygen scavengers, catalase (for H2O2) and n-propyl gallate (for hydroxyl radical) to the lysates also inhibited the fragmentation. When the purified Rubisco from wheat leaves was exposed to a hydroxyl radical-generating system comprising H2O2, FeSO4 and ascorbic acid, the LSU was degraded in the same manner as observed in the chloroplast lysates. The results suggest that the large subunit of Rubisco was directly degraded to the 37-kDa fragment containing the N-terminal region and the 16-kDa fragment containing the C-terminal region of the LSU by active oxygen, probably the hydroxyl radical, generated in the lysates of chloroplasts.

Ultraviolet‐B‐induced DNA lesions and their removal in wheat (Triticum aestivum L.) leaves

ABSTRACTMonoclonal antibodies were used in an enzyme‐linked immunosorbent assay (ELISA) to detect the induction and removal of cyclobutane pyrimidine dimers (CPDs) and (6‐4) photoproducts in DNA isolated from ultraviolet B (UV‐B)‐exposed primary wheat (Triticum aestivum L. cv. Chinese Spring) leaf tissue. The accumulation of lesions in the primary leaves of 6‐d‐old wheat seedlings was followed during the exposure of the leaf to an approximate dose of 3.6×10−1 W m−2 UV‐B (Caldwell weighting). Significant increases in the levels of both CPDs and (6‐4) photoproducts were detected in wheat leaves exposed to UV‐B in the absence of other light However, only an increase in (6‐4) photoproduct levels could be measured in wheat leaves exposed to the same UV‐B source in the presence of supplemental white light. The removal of CPD antibody binding sites in the DNA after irradiation was rapid under conditions of high light intensity in contrast to the removal of (6‐4) photoproduct antibody binding sites, which was significantly slower. The removal of CPDs appeared to be light dependent, this rate of removal decreasing with decreasing light fluences. The removal of (6‐4) photoproducts also appeared light dependent, but to a lesser extent than the removal of CPDs, under the conditions studied here. Gene expression in the primary wheat leaf was measured and showed an up‐regulation of chalcone synthase expression and a reduction in expression of chlorophyll a/b‐binding protein (cab) in response to supplementary UV‐B. No effect was seen on the expression of the other photosynthetic genes studied (the genes coding for the enzymes sedoheptu‐lose 1,7‐bisphosphatase and fructose 1,6‐bisphosphatase). Measurement of the levels of DNA lesions in this same tissue showed that the observed changes in gene expression accompanied the appearance of UV‐B induced lesions in the form of (6‐4) photoproducts in the wheat leaf genome.

Glucose feeding of intact wheat plants represses the expression of a number of Calvin cycle genes

ABSTRACTThe photosynthetic capacity and Rubisco levels of primary wheat leaves were reduced significantly in plants grown on media supplemented with 1 % glucose. Neither the chlorophyll content nor the growth rate of the primary leaf was affected by this treatment. In glucose‐fed plants a decrease in levels of mRNA encoding several Calvin cycle enzymes (fructose 1,6‐bisphosphatase, sedoheptulose 1,7‐bisphosphatase, phosphoglycerate kinase and Rubisco Small Subunit) was found to correspond to an increase in the internal levels of glucose. In control plants endogenous levels of glucose and sucrose were measured in leaf sections from the base to the tip of the primary leaf, which revealed a gradient of each of these sugars within the leaf, with the highest levels at the tip. Conversely, Calvin cycle transcripts were seen to decrease in the oldest tip sections. In glucose‐fed plants, the endogenous glucose levels were increased throughout the leaf but Calvin cycle RNA levels were most markedly reduced in the mid‐leaf section. These data indicate that the changing metabolic status of the cells from the base to the tip of the primary wheat leaf may play a role in the regulation of Calvin cycle gene expression during normal leaf development.

Changes to the Stoichiometry of Glycine Decarboxylase Subunits during Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.) Leaf Development

Changes in the levels of the four subunits of the mitochondrial enzyme glycine decarboxylase (EC 2.1.2.10) have been investigated during development in the 8 day old primary leaf of wheat (Triticum aestivum L.). Proteins were extracted from wheat leaf sections between the basal meristem and 8.5 centimeters. The individual glycine decarboxylase subunits were detected by Western blotting, using subunit-specific polyclonal antibodies, and quantified by laser densitometry. P, T, and H subunits showed similar developmental patterns along the leaf. All were below the level of detection up to 1.5 centimeters from the meristem, but then increased over the leaf length examined. In contrast, the increase in the L protein (lipoamide dehydrogenase) was more gradual, and levels in the youngest regions of the leaf were maintained at approximately 14% of those at 8.5 centimeters. In a complementary study, levels of the four subunits in light-grown leaf tissues were compared to those in etiolated leaves from wheat and pea (Pisum sativum L.), using the activity of the mitochondrial marker enzyme fumarase as the basis for comparison. For both wheat and pea, levels of P, T, and H proteins in etiolated tissues were between 25 and 30% of those in lightgrown tissue. However, in etiolated tissues L protein was present at levels of 60 to 70% of that in light-grown tissues. The results indicate that discrete mechanisms may control the synthesis of L, as compared to P, T, and H proteins.

Investigation of Galactolipid Turnover in Mature Primary Leaves of Wheat ( Triticum aestivum L.)

Summary Segments of attached primary leaves of wheat were labelled with either [ 14 C]urea or Na[l- 14 C]acetate, and label was measured in the major classes of membrane lipids over several days. Urea label is rapidly incorporated into soluble sugars, and heavily labels the galactose moiety of the galactolipids, while acetate label is directed predominantly into fatty acyl chains. PC was the most rapidly labelled lipid, but its activity declined asymptotically during the chase. The galactolipids were labelled more slowly, but continued to accumulate 14 C throughout the experiment. The persistance of label in leaf MGDG was assessed by examining the possible fluxes of label from PC, or to DGDG, in addition to the changes in radioactivity of the MGDG pool. The results gave no indication that any galactolipid label was lost through either cat-abolism of whole lipid molecules or exchange of fatty acids on the galactolipid backbone.

Does the Loss of Leaf Chlorophyll during Senescence of Primary Wheat Leaf Arise Due to Loss in Chloroplast Number or Chlorophyll Content?

Summary Relation between the chlorophyll loss on unit fresh mass basis and the loss of absorbance maximum at 678 nm of the chloroplast absorption spectrum during senescence was investigated in detached wheat leaves. The measurements of absorption spectrum in all assay intervals were made while maintaining nearly the same number of chloroplasts. We observed a close correlation between the extent of loss in chlorophyll content expressed on unit leaf fresh mass basis and the loss in the absorbance at 678 nm of chlorophyll a red maximum, computed per unit chloroplast basis, during leaf senescence. It is argued that senescence associated loss of chlorophyll during leaf senescence is due to uniform extent of loss in chlorophyll in all chloroplasts and not due to loss in chloroplast number.

Endopeptidase and Carboxypeptidase Enzymes of Vacuoles Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat Seedlings

Summary Vacuoles prepared from mesophyll protoplasts of the primary leaf of wheat ( Triticum aestivum L. cv. Egret) contain carboxypeptidase (CP) and endopeptidase (EP) activity. CP activity was optimal at pH 5.2. The most preferred substrate was Z-Phe-Ala and activity against a wide range of other substrates was dependent on the C-terminal amino acid. Z-dipeptides with proline in any position were not hydrolysed. CP activity was inhibited by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, sodium dodecyl sulfate (SDS) and 4-hydroxymercuric benzoic acid. Thus the CP enzyme from the wheat leaf is a Serine Carboxypeptidase (EC 3.4.16). EP activity, assessed by following the degradation of the Large Sub Unit (M r 53.1 kD) of Ribulose 1,5-Bisphosphate Carboxylase (RuBPCase) by SDS-PAGE, was optimal at pH 5.2 (-SDS) and 5.5 (+SDS). The overall rate of degradation of the Large Sub Unit (LSU) was increased about 2.6-fold by the addition of 0.2 % SDS to the assay medium. In the absence of SDS there were only three prominent polypeptide breakdown products, M r 52.1, 50.9 and 50.9 kD. The 50.9 and 50.1 kD products appeared resistant to further hydrolysis, whilst the 52.1 kD product was eventually degraded. The presence of SDS increased the heterogeneity of the breakdown products, polypeptides with M r 51.8, 44.4, 43.6, 30.3 and 29.5 kD were especially prominent. The major portion of the vacuolar EP activity was considered to belong to the Thiol proteinase group (EC 3.4.22), although indirect evidence was obtained for two other EP activities. There appeared to be little co-operation between the EP and CP activities in the degradation of RuBPCase.

Characteristics of a β-Galactosidase Associated with the Stroma of Chloroplasts Prepared from Mesophyll Protoplasts of the Primary Leaf of Wheat

Chloroplasts prepared from mesophyll protoplasts of the primary leaf of wheat (Triticum aestivum L. cv Egret) contain about 50% of the cellular beta-galactosidase (EC 3.2.1.23) activity. More than 80% of this activity is associated with the stroma and most of the remainder, although tightly bound to the thylakoids, can be washed free with sodium pyrophosphate. The vacuole contained about 20% and the remaining enzyme was presumed to be cytoplasmic or associated with one of the other organelles. Both the vacuolar and chloroplast enzymes were capable of releasing galactose from the galactolipid monogalactosyldiacylglycerol. Apart from their distinct locations within the cells, we conclude that the enzymes are different because they differed with respect to assay pH-optimum, comparative activity against the synthetic substrates phenyl-beta-d-galactoside, 4-methylumbelliferyl-beta-d-galactoside, 6-bromo-2-naphthyl-beta-d-galactoside, the disaccharide lactose, and the inhibitors d-galactose and d-galactono-1,4-lactone.

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.)

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.

Isolation and Some Properties of an Aminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

The isolation and characterization of the AP1 form of aminopeptidase (EC 3.4.11.) previously identified (Waters, Dalling 1979 Aust J Plant Physiol 6: 595-606) in the primary leaves of wheat (Triticum aestivum L. cv Egret) seedlings is reported. The enzyme shows a high preference for a substrate which contains an aromatic side chain, whether this be either a synthetic beta-naphthylamide or a peptide substrate. Maximum activity with both types of substrates occurred around pH 7.6. The stability of AP1 was reduced by exposure to high pH and by incubation at temperatures above 20 degrees C in the absence of substrate. AP1 was inhibited by the metal chelators bathocuproine and bathophenanthroline and the sulfhydryl group inhibitors p-chloromercuribenzoate and N-ethylmaleimide. The molecular weight, estimated by gel filtration, was 57,000. The K(m) value for activity against the synthetic substrate Phe-beta-NA (0.20 millimolar) was slightly lower than that for Phe-Phe (0.50 millimolar) although the enzyme activity against peptide substrates was considerably greater than with Phe-beta-NA.

Relation between Ribulose Bisphosphate Carboxylase Content and Chloroplast Number in Naturally Senescing Primary Leaves of Wheat

Relation between Ribulose Bisphosphate Carboxylase Content and Chloroplast Number in Naturally Senescing Primary Leaves of Wheat

Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the beta-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp-Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity.

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat. II. Main Phosphorylated Fractions

Seedlings of wheat (Triticum durum, cv. Balcarceno-INTA) were water-stressed in darkness with 20% polyethylene glycol (PEG) 6000 or 0-3 M mannitol added to the root medium. At different times and up to a total of 36 h of treatment the coleoptiles and primary leaves were cut and analysed for acid soluble-P, lipid-P, protein-P, alkali-stable organic-P, and nucleic acid-P. AU the phosphorylated fractions were expressed on 100 mg of total coleoptile and primary leaves dry wt. Acid soluble-P (14-6 /miol) accounted for most of the phosphorus. The lipid-P fraction remained at an unchanged level (6-2 /nnol/100 mg dry wt) in control or in mildly osmotic stressed shoots of seedlings over the period of treatment. The protein-P represented only 2-4% (0-8 //mol) of the total phosphorus found in coleoptiles and primary leaves. The alkali-stable organic-P and nucleic acid-P fractions represented 22-2% (7-2 fimai) and 11-1% (3-6 fmo\) of the total phosphorus content of shoots, respectively. All the phosphorylated fractions, excepting the lipid-P fraction, underwent a significant {P < 0-025) fall over periods of up to 36 h of wheat seedlings growth in darkness. A common characteristic found in all the phosphorylated fractions was the fact that there were not significant differences between control and 20% PEG 6000 or 0-3 M mannitol treated seedlings during 36 h of treatment and growth in darkness. However, when seedlings were pulse-labelled with 32P during imbibition, some effects of the osmotic stressants on several fractions could be seen. Specific radioactivity fell in acid soluble-P of control shoots, but increased in 20% PEG 6000 and in 0-3 M mannitol treated seedlings. Radioactive phosphorus was not found in the alkali-stable organic-P fraction. Lipid-P, nucleic acid-P, and protein-P fractions increased their specific activities during 36 h of shoot growth in control plants. Such 20% PEG 6000 and 0-3 M mannitol restricted this increase during the same period of time, the former being more active. Possible implications of phospholipid and phosphoprotein turnover in relation to water stress are discussed.

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat: I. EFFECTS ON SIZE, WEIGHT, TOTAL PROTEIN, DNA, AND PHOSPHORUS

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat: I. EFFECTS ON SIZE, WEIGHT, TOTAL PROTEIN, DNA, AND PHOSPHORUS

Specificity of induction of the lignification response in wounded wheat leaves

A range of abiotic treatments reported to induce phytoalexin accumulation in various plants failed to induce lignification in a wounded primary wheat leaf system. The inducers included antimetabolites, metabolic inhibitors, DNA-intercalating compounds, basic polypeptides, oxidising and reducing agents, halogen anions, heavy metal ions and u.v. irradiation. Only mercuric ions elicited a response. A range of fungi, including species normally of saprophytic habit, species pathogenic on plants other than wheat, and wheat pathogens all elicited lignification. Yeasts and bacteria, unlike the mycelial fungi, were poor elicitors of the response. Thus lignification appeared to be induced almost exclusively by fungi, unlike phytoalexin production which can also be elicited by a wide range of abiotic treatments.