Non-photosynthetic Tissues Research Articles

Growth and Photosynthetic Responses of Spinach to Salinity: Implications of K+ Nutrition for Salt Tolerance

To compare the effects of K+ under high and low salinity, spinach plants (Spinacia oleracea) were grown in nutrient solutions containing either 50 mM NaCl (low salinity) or 250 mM NaCl (high salinity), with a diurnal regime of 10 h light (~300 μmol photons m-2 s-1, 23°C) and 14 h dark (15°C). At each level of salinity, the nutrient KCl concentration was 0.01, 0.1, 1 or 10 mM. The plant and shoot biomass was greater at low salinity than high salinity and increased with the logarithmic increase in nominal K+ concentrations supplied to the roots. Plant and shoot growth were related to the K+ uptake into the leaves, with leaves having a higher K+ content under low salinity than high salinity. Variation of the K+ content in the leaves, induced by the combinations of nutrient KCl concentrations with high or low salinity, were accompanied by changes in the photosynthetic capacity at light- and CO2-saturation per unit leaf area; there was a greater decrease in photosynthetic capacity with decreasing K+ supply to the roots under high salinity than under low salinity. The photosynthetic capacity was in turn highly correlated with the contents of cytochrome f and ATP synthase per unit leaf area. Under conditions of high salinity and low K+ supply, a reduction in the quantum yield of oxygen evolution also occurred, due to malfunction of photosystem II and, apparently, an increased proportion of light absorbed by non-photosynthetic tissue. The decreases in photosynthetic capacity and quantum yield partly account for the lower plant and shoot biomass at high salinity and low nutrient KCl concentrations. Our results suggest strongly that there are higher K+ requirements for shoot growth under high than low salinity conditions, and that high concentrations of Na+ in the leaves may help to maintain turgor, but cannot substitute for adequate K+ levels in the leaves, presumably because K+ is specifically required for protein synthesis. Increasing the K+ supply at the roots can ameliorate reductions in plant and shoot biomass imposed by an increase in salinity.

Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.

CuZn-Superoxide Dismutases in Rice: Occurrence of an Active, Monomeric Enzyme and Two Types of Isozyme in Leaf and Non-Photosynthetic Tissues

CuZn-Superoxide Dismutases in Rice: Occurrence of an Active, Monomeric Enzyme and Two Types of Isozyme in Leaf and Non-Photosynthetic Tissues

Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus.

Carotenoid pigments are essential for the protection of both photosynthetic and non-photosynthetic tissues from photooxidative damage. Although carotenoid biosynthesis has been studied in many organisms from bacteria to higher plants, little is known about carotenoid biosynthetic enzymes, or the nature and regulation of the genes encoding them. We report here the first DNA sequence of carotenoid genes from any organism. We have determined the complete nucleotide sequence (11,039 bp) of a gene cluster encoding seven of the eight previously known carotenoid genes (crtA, B, C, D, E, F, I) and a new gene, designated crtK, from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium. The 5' flanking regions of crtA, I, D and E contain a highly conserved palindromic sequence homologous to the consensus binding site for a variety of prokaryotic DNA-binding regulatory proteins. This putative regulatory palindrome is also found 5' to the puc operon, encoding the light-harvesting II antenna polypeptides. Escherichia coli-like sigma 70 promoter sequences are located 5' to crtI and crtD, suggesting for the first time that such promoters may exist in purple photosynthetic bacteria. The crt genes form a minimum of four distinct operons, crtA, crtIBK, crtDC and crtEF, based on inversions of transcriptional orientation within the gene cluster. Possible rho-independent transcription terminators are located 3' to crtI, B, K, C and F. The 3' end of crtA may overlap transcription initiation signals for a downstream gene required for bacteriochlorophyll biosynthesis. We have also observed two regions of exceptional amino acid homology between CrtI and CrtD, both of which are dehydrogenases.

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts - a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP + reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein ( E m = −440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Enzymes of Glucose Oxidation in Leaf Tissues

The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C(3)-plants, pea and leek, and two C(4)-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.

Gene Expression in Developing Wheat Endosperm

The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding alpha/beta- and gamma-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages of development was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically.

Light-induced increases in the glycine decarboxylase multienzyme complex from pea leaf mitochondria

The rates of mitochondrial glycine oxidation estimated by CO 2-release and glycine-bicarbonate exchange activities in fully greened tissues are approximately 10 times greater than those of etiolated pea leaves and potato tuber mitochondria. The release of CO 2 from glycine in intact mitochondria isolated from dark-grown and nonphotosynthetic tissues was sensitive to inhibitors of mitochondrial electron transport, glycine transport, and glycine decarboxylase activities. The CO 2-release and glycine-bicarbonate exchange activities in crude mitochondrial protein extracts from light-grown versus dark-grown tissues exhibited light/dark ratios of 12 and 21, respectively. This suggests that the differences in capacity to oxidize glycine reside with the glycine decarboxylase enzyme complex itself. The complex is composed of four subunit enzymes, the P, H, T, and L proteins, which can be isolated individually and reconstituted into the active enzyme. The activities of P and T proteins were at least 10 times higher in fully greened pea leaves than in the etiolated tissue, while the H and L protein activities were four times higher in these same tissues. The levels of P and T proteins detected immunochemically were substantially lower in total mitochondrial extracts prepared from leaves of dark-grown pea seedlings. Labeling of whole pea seedlings and in vitro protein synthesis with isolated mitochondria indicated that the entire glycine decarboxylase enzyme complex is cytoplasmically synthesized and therefore encoded by the nucleus. Polypeptides synthesized from total leaf polyadenylated mRNA isolated from leaves of both the dark-grown and light-treated peas indicated the presence of P protein. This implies that translatable messages for this enzyme are present at some level throughout leaf development.

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30-40% of the measured rate of CO2 fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2-5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.

Specific heat shock proteins are transported into chloroplasts

We demonstrate that in three plant species-soybean, pea, and corn-certain nuclear-encoded heat shock proteins are transported into chloroplasts. In vitro translation products of poly(A)-RNA from control or heat-shocked plants were incubated with isolated intact pea chloroplasts and differences in the profile of imported proteins were analyzed. In all three species, abundant polypeptides between 21 and 27 kDa are present in the heat shock sample and absent in the controls. These polypeptides are protected from trypsin and chymotrypsin digestion after their import into chloroplasts and are recovered primarily with the soluble chloroplast protein fraction. Chloroplasts isolated from pea or corn leaves labeled in vivo at heat shock temperatures, but not at normal growth temperatures, contain the same polypeptides observed in vitro. Synthesis of the heat shock polypeptides can be inhibited in vivo by cycloheximide but not by chloramphenicol, further indicating they are products of cytoplasmic protein synthesis. The in vitro transport experiments demonstrate that synthesis of the chloroplast-localized heat shock proteins results from heat-induced accumulation of the corresponding poly(A)-RNAs. The same mRNAs are also produced in response to heat shock by a nonphotosynthetic tissue, the etiolated soybean hypocotyl.

Biomass, production and decomposition of a deep water seagrass, Halophila decipiens ostenf.

Beds of Halophila decipiens Ostenfeld, at depths between 15 and 27 m, were studied using a saturation diving facility (NULS-1: Hydrolab) in the Salt River submarine canyon off St. Croix, US Virgin Islands. Distribution, biomass, production and decomposition of the seagrass were studied during two missions in June 1983 and July 1984. Biomass of H. decipiens ranged from 5 to 12 g m −2 during summer months. Due to the rapid turnover of H. decipiens, biomass distribution was closely related to concurrently measured growth rates. Production estimates using oxygen production techniques or rhizome elongation were similar ranging between 100 and 500 mg C m −2 day −1. Decomposition of H. decipiens occurred rapidly, losing over 50% of its original weight in approximately 3 days. Under most conditions, weight loss from litter bags occurred more rapidly than nitrogen loss. H. decipiens exhibits a number of adaptations to a low ambient light environment, including a high ratio of leaf tissue to non-photosynthetic tissue, low leaf area index to reduce self-shading, high turnover leaf material and the ability to rapidly colonize sandy bottoms when light conditions are suitable.

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.

Biochemical Characterization of Chlorophyll-Free Mitochondria From Pea Leaves

Mitochondria from pea leaves were purified by centrifugation on a self-generated Percoll gradient which contained a linear gradient of polyvinylpyrrolidone-25 (0-10%, w/v). The chlorophyll content of the purified mitochondria was less than 1 �g per mg protein. All substrates were rapidly oxidized by these mitochondria, the rate of glycine oxidation being between 200 and 300 nmol O2 min-1 mg-1 protein, depending on the age of the leaves used. These rates did not vary significantly over a period of 20 h, provided NAD+ was supplied exogenously, when the mitochondria were stored on ice. Respiratory control, ADP/O ratios and outer membrane integrity (always more than 95%) were also maintained during storage. The phospholipid composition of the membranes from the leaf mitochondria was virtually identical to that of mitochondria from non-photosynthetic tissues although their lipid to protein ratio was slightly lower. The polypeptide pattern of the membranes from green leaf mitochondria and those from etiolated leaves and hypocotyls were also similar, but marked differences were observed between the matrix proteins from the different tissues. In particular, intensely stained bands at 94, 51,41 and 15.5 kDa which were present in the matrix of green leaf mitochondria were missing or present in much smaller quantities in the non-photosynthetic tissues. This difference was correlated with the ability of the mitochondria to oxidize glycine, suggesting that the four polypeptides may be associated with the glycine decarboxylase complex.

Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max

The aim of this work was to determine the relative contributions of ADPglucose and UDPglucose to starch synthesis in two non-photosynthetic tissues, the developing club of the spadix of Arum maculatum and suspension cultures of Glycine max. Rates of starch accumulation during growth are compared with estimates of the maximum catalytic activities in vitro of ADPglucose starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and UDPglucose starch synthase. The latter could only be measured at high concentrations (10–30 mM) of UDPglucose. Clubs of Arum and cells of Glycine contained 292 and 6.8 nmol UDPglucose per gram fresh weight, respectively. The corresponding figures for ADPglucose were 29 and 0.4. From the above data it is argued that in both Arum club and Glycine cells the activity of UDPglucose starch synthase is too low to make any quantitatively significant contribution to starch synthesis. The activities of ADPglucose starch synthase and pyrophosphorylase were high enough to mediate the observed rates of starch accumulation. It is suggested that starch synthesis in these tissues is via ADPglucose.

Role and location of NAD malic enzyme in thermogenic tissues of araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO 2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2- N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO 2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.

A comparison of the membrane composition of mitochondria isolated from spinach leaves and leaf petioles

Very pure preparations of mitochondria were obtained from spinach leaves and leaf petioles by a preparation procedure combining differential centrifugation, phase partition and Percoll density gradient centrifugation. These preparations were used for measurements of cytochrome content, lipid content and distribution of proteins between the membranes and the matrix fraction. Leaf and petiole mitochondria contained qualitatively the same cytochromes as measured by difference spectroscopy. On a protein basis leaf mitochondria contained only half the amount of cytochromes compared to petiole mitochondria. Leaf mitochondria had also a somewhat lower lipid/protein ratio than petiole mitochondria. In leaf mitochondria a relatively higher proportion of the protein was in the matrix fraction compared to petiole mitochondria. It is concluded that leaf mitochondria have less respiratory chains in relation to other membrane components compared to petiole mitochondria. The differences in composition are discussed with relation to metabolic differences between mitochondria in photosynthetic tissue and non-photosynthetic tissue.