Non-green Tissues Research Articles

In vivo and in vitro characterization of protein interactions with the dyad G-box of the Arabidopsis Adh gene.

Expression of the alcohol dehydrogenase (Adh) and ribulose-1,5-bisphosphate carboxylase small subunit (RbcS) genes of higher plants is cell-type-specific and environmentally inducible. However, the tissues in which these two genes are expressed, their modes of induction, and their protein functions are quite distinct. Adh is expressed in non-green tissue, induced by anaerobiosis, and repressed in leaves. RbcS is only expressed in green tissue. An 8-base pair G-box element (5'-CCACGTGG-3') is associated with light-induced expression of RbcS and chalcone synthase. The same sequence is also present in the 5'-flanking region of Arabidopsis thaliana Adh, and this sequence is associated with a trans-acting factor in vivo. We report here that in vitro Adh G-box binding activity is present in crude whole cell extracts of both cell culture and leaves of Arabidopsis. The authenticity of in vitro Adh G-box binding is supported by in vivo and in vitro dimethylsulfate footprinting. A clear in vivo Adh G-box footprint occurs in cell cultures, but comparable in vivo binding to the Adh G-box does not occur in leaves. Therefore, there does not appear to be a direct correlation between the presence of the G-box factor in a tissue and its binding to the Adh G-box.

The estimation of l-phenylalanine ammonia-lyase shows phenylpropanoid biosynthesis to be regulated by l-phenylalanine supply and availability

l-Phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) is the first committed enzyme in the pathway leading to phenylpropanoid biosynthesis in higher plants. PAL catalyses the conversion of l-phenylalanine to t-cinnamic acid with the elimination of ammonia. Standard methods for determination of PAL activity in both green and non-green tissues were found to lead to measurements of both l-phenylalanine amino-transferase (PAT, E.C. 2.6.1.1) and PAL activities together. The accurate estimation of PAL activity alone, necessitated the inhibition of PAT by a specific inhibitor of PAT activity, l-aspartic acid. The influence of PAT on the kinetics of PAL activity may explain (i) the diverse properties that have been attributed to PAL and (ii) the controversies regarding the control mechanism underlying the regulation of PAL activity. Evidence is presented for the regulation of phenylpropanoid biosynthesis via substrate supply and availability as opposed to feedback inhibition, during phaseollin production and hypersensitive necrosis in Phaseolus vulgaris.

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.

Sorbitol Metabolism and Sink-Source Interconversions in Developing Apple Leaves

In apple (Malus domestica Borkh.) sorbitol is the primary product of photosynthesis, the major translocated form of carbon, and a common fruit constituent and storage compound. Previous work on sorbitol metabolism has revealed a NADPH-dependent aldose 6-phosphate reductase (A6PR) in green tissues, and a NAD-dependent sorbitol dehydrogenase in nongreen tissues. Results here show a decrease in sorbitol dehydrogenase activity and an increase in A6PR activity as leaves developing in the spring undergo the transition from sink to source. Sorbitol dehydrogenase activity reached a minimum as A6PR peaked. These changes were related to increases in leaf carbohydrate levels, especially sorbitol, and to increases in rates of net photosynthesis. Studies conducted in the autumn on senescing leaves also showed changes in enzyme activites, leaf carbohydrate levels, and photosynthesis. At this time, however, sorbitol dehydrogenase increased in specific activity, whereas A6PR activity, leaf carbohydrates, and photosynthetic rates all decreased substantially. Other experiments showed differences in the ability of young and mature leaves to metabolize sorbitol and in the distribution of sorbitol enzymes in leaves at transitional developmental stages. The results suggest that sorbitol metabolism in apple is tightly controlled and may be related to mechanisms regulating partitioning or source and sink activity.

Effects of Toxin fromHelminthosporium saccharion Nongreen Tissues and a Reexamination of Toxin Binding

Effects of Toxin from<i>Helminthosporium sacchari</i>on Nongreen Tissues and a Reexamination of Toxin Binding

Effects of Toxin fromHelminthosporium saccharion Nongreen Tissues and a Reexamination of Toxin Binding

Effects of Toxin from<i>Helminthosporium sacchari</i>on Nongreen Tissues and a Reexamination of Toxin Binding

Phospholipid composition of chlorophyll-free mitochondria isolated via protoplasts from oat mesophyll cells.

Mitochondria were isolated from oat primary leaves via mesophyll protoplasts and subjected to phospholipid analysis. In mesophyll cells mitochondria account for only small proportions of cellular phospholipids (in the order of 5%) and proteins (in the order of 2%). Contamination by lipids from other membranes was insignificant as indicated by the absence or very low levels of chlorophyll, galactolipids and steryl glycosides. The absence of 3-trans-hexadecenoic acid in phosphatidylglycerol from mitochondria of green cells serves an an additional criterion of purity. The phospholipid mixture extracted from these mitochondria resembles phospholipids in mitochondria from non-green tissues regarding composition as well as fatty acid profiles. Therefore, mitochondria maintain a rather constant lipid profile and in contrast to plastids do not respond at this level to differences in the physiological status of their housing cell. Palmitic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine is primarily localized at the C-1 position of the glycerol moiety. Two enzymatic activities so far not described in mitochondria, formation of acylgalactosyl diacylglycerol and hydrolysis of acyl-CoA, were found in the purified mitochondrial fraction.

Phytochrome in Callus Tissue Cultures of Cruciferae

Summary Phytochrome content in non-green callus tissue cultures from several species of the Cruciferae has been assayed in vivo . The highest measurable amount of phytochrome occurred in callus tissue from Sinapis alba . The magnitude of phytochrome content appears not to depend on the type of callus, i. e. whether it is a friable or a compact one. Comparable amounts of phytochrome in starting material and the appropriate callus tissue were observed only if the callus was derived from mature organs. Phytochrome dark transformations were considerably faster in friable than in compact callus tissue. Reappearance of photoconvertible phytochrome in darkness following destruction in red light extended over a period of about 5 days until the level of the dark control value had been regained.

Two separate photoreceptors control hypocotyl growth in green seedlings

PHOTOCONTROL of elongation growth has been extensively studied in dark-grown (etiolated) seedlings for some time. However, only relatively recently has detailed attention been given to green (de-etiolated) plants. One reason for this is that the photoreceptor, phytochrome, can easily be detected spectrophotometrically in non-green tissues but not in tissue containing chlorophyll. There is agreement that the photoreceptor operates in its low-energy mode (low irradiances or fluence rates, with red/far-red reversibility) in green seedlings as well as in dark-grown, non-green seedlings. In its high-energy mode (high irradiances and time dependency), exemplified by the action of prolonged irradiation with far-red, phytochrome might act only in etiolated seedlings1–3. Photoregulation of stem extension in etiolated and de-etiolated seedlings is also achieved by blue light (400–500 nm), but it is still not clear if a photoreceptor other than phytochrome is responsible for this effect. Kinetic studies on etiolated seedlings suggest the operation of a separate blue-responsive photosystem2. Notwithstanding this, it has recently been suggested that in green seedlings blue-light control of hypocotyl extension is due solely to absorption by phytochrome4. This claim, together with the evidence that changes in phytochrome photoequilibria can exert profound effects upon stem extension, may lead one to believe that this is the major photoreceptor controlling elongation growth in green plants. We report here, however, work that strongly indicates that two separate pigment systems control hypocotyl extension in green seedlings—phytochrome and a separate blue-lightreceptor. Both systems produce the same response—inhibition—but we have found it possible to separate their effects by examining the times at which the response commences.

Subcellular Localization of Isocitrate Lyase in Nongreen Tissue Culture Cells

Density gradient centrifugation and electron microscopy were used to establish that isocitrate lyase present in Rosa cv. Paul's Scarlet cells was located in the mitochondria and not other membrane fractions. The enzyme may be important in glycine and serine synthesis. A comparison between the enzymic activity of isocitrate lyase and the amount of glycine and serine synthesized during logarithmic growth indicated that the activity was great enough to account for all of the carbon entering these amino acids during that stage of growth.

Kinetin-induced Changes in δ-Aminolevulinic Acid Dehydratase of Tobacco Callus

Kinetin-induced changes in dry weight, soluble protein content, delta-aminolevulinic acid dehydratase activity, and chlorophyll content of two clones of Nicotiana tabacum L. callus were studied. Kinetin brought about a marked increase in the delta-aminolevulinic acid dehydratase activity of nongreen tissue just before induction of greening. The experimental data suggested a possible induction of specific chloroplast protein(s) during the kinetin-induced greening of nongreen tobacco tissue. Kinetin caused a decline in the delta-aminolevulinic acid dehydratase activity and chlorophyll content of the green callus used in the present study.

A Comparison of Nitrite Reductase Enzymes from Green Leaves, Scutella, and Roots of Corn (Zea mays L.)

Nitrite reductase from green leaves of corn (Zea mays L.) is eluted from a diethylaminoethyl-cellulose column in one peak of activity by a chloride gradient, while nitrite reductase from scutellum tissue is resolved into two peaks of activity, apparently representing two forms of the enzyme NiR1 and NiR2. One of these (NiR2) elutes at the same concentration of chloride as the leaf nitrite reductase. Roots and etiolated shoots also exhibited both forms of the enzyme, however, lesser amounts of NiR1 is extractable from these tissues than from scutellum. Comparison of green leaf nitrite reductase with NiR2 from scutellum tissue shows similar or identical properties with respect to molecular weight, isoelectric point, electron donor requirements, inhibition properties, pH optima, thermal stability, and pH tolerance. The significance of these similarities in relation to probable differences in the biochemical mechanism of nitrite reduction between leaf and scutellum tissues is discussed. Although ferredoxin is considered, with some reservations, to be the electron donor for nitrite reductase in green tissue, the reductant for nongreen tissue is not known. The possibility that nitrite reductases from green and non-green tissues uses the same electron donor, in vivo, is considered.

Microbody Enzymes and Carboxylases in Sequential Extracts from C4 and C3 Leaves

A seven-step sequential grinding procedure was applied to leaves of Atriplex rosea, Sorghum sudanense, and Spinacia oleracea to study the distribution of carboxylases and microbody enzymes. In the extracts from C(4) species there were 7- to 10-fold reciprocal changes in specific activities of ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase. No such changes occurred in sequential extracts from spinach. No inhibitors of ribulose-1, 5-diphosphate carboxylase were detected when the mesophyll extracts of Sorghum were assayed together with spinach extracts. These results reaffirm the conclusion of others that phosphoenolpyruvate carboxylase is largely confined to the mesophyll in these species and ribulose-1, 5-diphosphate carboxylase to the bundle sheath. The specific activities of glycolate oxidase and hydroxypyruvate reductase in bundle sheath extracts were two to three times those in mesophyll fractions. Catalase behaved similarly in Atriplex rosea but in Sorghum the specific activity was virtually the same in all fractions. From the relative amounts of these enzymes present, and comparison with the data obtained from spinach, it is concluded that typical leaf peroxisomes are present in the bundle sheaths of both C(4) species and in the mesophyll of Atriplex rosea. The relative enzyme activities in the mesophyll of Sorghum suggest that the microbodies there are of the non-specialized type found in many nongreen tissues. The activities of the microbody enzymes in the bundle sheath of Sorghum seem quite inadequate to support photorespiration.

Growth substance requirements and major lipid constituents of tissue cultures of Euphorbia esula and E. cyparissias

The root tissues of Euphorbia esula and E. cyparissias form callus on chemically defined medium. Both species require an exogenous supply of auxin for growth, but the appearance and color of the tissue and their responses to kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) are different. The tissue growth is more satisfactory with α-naphthaleneacetic acid (NAA) than with 2,4-D, IAA, or 4-amino-3,5,6-trichloropicolinic acid (picloram). Gibberellic acid has no effect. The callus tissues of E. esula become intensely green under light but are not autotrophic.Triglycerides, palmitic acid, and β-sitosterol are the major lipid constituents of the callus tissue of E. esula. Chromatographic analysis reveals no significant differences in the composition of extracts from the non-green and green tissues. Long-chain aldehydes, alcohols, and triterpenes found in the plant are not detected in the cultures.

The Isolation of Proplastids from Roots of Vicia faba

To our knowledge most living cells in higher plants contain plastids. In nongreen tissues such as primary roots, the plastids have been identified by the rather ambiguous name These proplastids are generally described as rather simple organelles, bounded by a double envelope and containing only a few internal membranes with a limited number of invaginations of the inner membrane of the plastid envelope (4). Ribosomes also apparently occur in proplastids (6) and depending on where the proplastids occur in the root the organelles may contain starch grains (3, 6). Proplastids in some roots are somewhat more complex in that they store protein and tubular complexes of membranes occur (6). However, the prefix pro might imply that proplastids have limited functional significances in the growth, development, and metabolism of root cells and are primarily latent progenitors of other types of plastid of more functional significance in the plant. There is almost no direct information on the biochemical properties and functional roles of proplastids in tissues such as roots. The present paper reports a method for the isolation of proplastids from roots of Vicia faba. Electron microscopical examination of the isolated fraction revealed that it contained numerous proplastids in an excellent state of preservation, substantially freed from cellular contamination, and thus would provide good starting material for the investigation of the properties of proplastids.

Effects of Sucrose and Kinetin on Growth and Chlorophyll Synthesis in Tobacco Tissue Cultures

Investigations were carried out on the effects of various combinations of sucrose and kinetin concentrations on growth and chlorophyll production in a green and a nongreen clone of pith callus of Nicotiana tabacum L. It was found that 2 milligrams per liter or higher amounts of kinetin induced greening in the nongreen tissue. The observations suggested that growth of the callus and synthesis of chlorophyll and soluble protein are controlled by relative concentrations of sucrose and kinetin in the medium. Kinetin was found to be inhibitory for chlorophyll synthesis in the green callus.

Specificity of Cycloheximide in Higher Plant Systems

Although cycloheximide is extremely inhibitory to protein synthesis in vivo in higher plants, the reported insensitivity of some plant ribosomes suggests that it may not invariably act at the ribosomal level. This suggestion is reinforced by results obtained with red beet storage tissue disks, the respiration of which is stimulated by cycloheximide at 1 microgram per milliliter. Inorganic ion uptake by these disks is inhibited by cycloheximide at 1 microgram per milliliter while the uptake of organic compounds, by comparison, is unaffected. Ion uptake by all nongreen tissues tested is inhibited by cycloheximide, but leaf tissue is unaffected, indicating that the ion absorption mechanism in the leaf may differ fundamentally from that in the root. It is concluded that cycloheximide can affect cellular metabolism other than by inhibiting protein synthesis and that the inhibition of ion uptake may be due to disruption of the energy supply.

Studies on Respiratory Enzymes in Rice Kernel

The flavoprotein highly purified from rice embryo has been shown to have not only NADPH diaphorase activity but also ferredoxin NADP reductase and pyridine nucleotide transhydrogenase activities. The enzyme catalyzed the reduction of various artificial electron acceptors such as 2, 6-dichlorophenol indophenol, potassium ferricyanide, and quinone with the strict requirement for NADPH as the electron donor. The pH optimum and the affinities of the enzyme for 2, 6-dichlorophenol indophenol and NADPH have been determined. Close similarities of enzymatic properties were found between the rice embryo flavoprotein and the spinach chloroplast NADPH diaphorase. The physiological role of the enzyme is discussed in relation to the cellular metabolism in nongreen tissues of higher plants.

NOTE ON PHOTOSYNTHETIC ACTIVITY IN SEEDS OF THE SPIDER LILY

THE IDEA of a foliar origin of the ovule is of long standing in botany, and perhaps as a corollary it may be noted that there are many evidences of the persistence of chlorophyllous tissue in seeds. For the most part this persistence appears juvenile within translucent ovaries, disappearing with the development of opacity in the enveloping structures; yet it may be assumed that the elaborated carbohydrates contributed to the development of seed structures. Flint (1938) indicated that the quality of light absorbed by the chlorophyllous tissues of maturing seeds and the enclosing carpels appeared related to the property of light-sensitivity in seeds. Such phenomena as the greening of seedlings in light and the relation of quality of light to the germination of light-sensitive seeds as reported by Flint and McAlister (1937) involve chlorophyll in macroscopically non-green tissue, yet may be cited as further instances of the persistence of chlorophyll. Ordinarily the principal source of organic reserves making possible the development of the ovule in the higher plants is the chlorophyllous tissue of the parent plant. Compared with this source the photosynthetic activities of the embryo or of the accessory surrounding tissues are meager and transitory. In the seeds of the spider lily, however, and probably in seeds of a similar nature, there is normally a delayed growth of the very young embryo accompanied by a development of accessory tissues (Whitehead and Brown, 1940). There is no obvious concentration of organic reserves for the maintenance of the embryo and for its support during germination. Instead, such organic reserves as are supplied by the parent plant through the funiculus are utilized in the extensive elaboration of the accessory tissues. The integuments develop into a somewhat elaborate chlorophyllous tissue with sto1 Received for publication November 9, 1942. mates, underlain with parenchyma and vascular strands. Such a development carries with it the implication of functional activity for the chlorophyllous tissue, but the direct demonstration of photosynthetic activity is rendered obscure by the failure of the tissue to store starch and by the absence of dehydration and an attendant inactivity of a partially-developed embryo. Respiration may be assumed to be active in the developing embryo, contributing further to the utilization of carbohydrates. The developing embryos push through the seedcoats without the ordinary relation to soil and moisture, and even the formation of germling plants may take place on a laboratory table. It was found by Flint (1943), however, that immersion in water effected an abrupt arresting of the development of the embryo. Seeds thus immersed for ten months were found to have an unimpaired viability, however, resuming germination in the normal time and manner when removed from water. This discovery suggested the possibility of prolonging photosynthetic activity while embryo development was arrested and led to the experiments here reported. MATERIALS AND METHODS.-About ten quarts of seeds of the spider lily, Hymenocallis occidentalis, were collected on May 29, 1942. These were separated into groups on the basis of the weights of the individual seeds, as 4 to 5 gms., 5 to 6 gms., etc. The seeds selected were all green and firm, without injuries. Comparisons were designed to involve seeds of the same groups insofar as was possible. The selected lots of seeds were weighed, immersed in water for twenty-four hours, dried on moist paper towels, and re-weighed. They were then placed under the experimental conditions, which included immersion in series in water in large-bore pyrex glass tubes in the laboratory near a south window. Light was excluded from some of the tubes