Distribution of alpha-Galactosidase in Cucurbita pepo

The distribution of α-galactosidase (α-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([α-1-6-0-galactopyranosyl]_n sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of α-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total α-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant.     

The Heterogeneity of Structural and Functional Photosynthetic Characteristics of Mesophyll Chloroplasts in Various Parts of Mature or Senescing Leaf Blade of Two Maize (Zea Mays L.) Genotypes

Differences in ultrastructural parameters of mesophyll cell (MC) chloroplasts, contents of photosynthetic pigments, and photochemical activities of isolated MC chloroplasts were studied in the basal, middle, and apical part of mature or senescing leaf blade of two maize genotypes. A distinct heterogeneity of leaf blade was observed both for structural and functional characteristics of chloroplasts. In both mature and senescing leaves the shape of MC chloroplasts changed from flat one in basal part of leaf to nearly spherical one in leaf apex. The volume density of granal thylakoids decreased from leaf base to apex in both types of leaves examined, while the amount of intergranal thylakoids increased in mature leaves but decreased in senescing leaves. The most striking heterogeneity was found for the quantity of plastoglobuli, which strongly increased with the increasing distance from leaf base. The differences in chloroplast ultrastructure were accompanied by differences in other photosynthetic characteristics. The Hill reaction activity and activity of photosystem 1 of isolated MC chloroplasts decreased from leaf base to apex in mature leaves. Apical part of senescing leaf blade was characterised by low contents of chlorophyll (Chl) a and Chl b, whereas in mature leaves, the content of Chls as well as the content of total carotenoids (Car) slightly increased from basal to apical leaf part. This was reflected also in the ratio Chl (a+b)/total Car; the ratio of Chl a/b did not significantly differ between individual parts of leaf blade. Both genotypes examined differed in the character of developmental gradient observed along whole length of leaf blade.     

Role of organic acids in the formation of the ionic composition in developing glycophyte leaves

The ionic composition in the leaves of some glycophyte plants (Phaseolus vulgaris L., Lycopersicon esculentum L., and Amaranthus cruentus L.) was studied during leaf development. Plants were grown in a stationary hydroponic culture; a growth medium contained equimolar concentrations of inorganic ions (NO ₃ ^? , Cl^?, SO ₄ ^2? , H₂PO ₄ ^? , K⁺, Ca²⁺, Mg²⁺, and Na⁺) equal to 5 mg-equiv./l for each ion. In the juvenile leaf, the main ions were K⁺ and water-soluble anions of organic acids represented mainly by di-and tricarboxylic acids in kidney bean and tomato and oxalic acid in amaranth. An increase in the total amount of organic anions, coinciding with the accumulation of bivalent cations, was registered in leaves of glycophytes during their development. Mature and senescing leaves of tomato and kidney bean accumulated mainly di-and tricarboxylic acid salts with the prevalence of Ca²⁺ ions. In amaranth leaves, the formation of water-insoluble (acid-soluble) oxalate pool containing Ca²⁺ ions (mature leaves) or Ca²⁺ and Mg²⁺ ions (senescing leaves) was registered. The priority role of the metabolism of organic acids in the formation of the ionic composition of glycophyte leaves during their development is discussed. It is supposed that the species-specific ionic composition of glycophyte leaves at different developmental stages is due mainly to the pattern of carbon metabolism causing the accumulation either of di-and tricarboxylic acids or oxalic acid.     

Free tryptophan and indole-3-acetic acid levels in the leaves and vascular pathways of Ricinus communis L.

Levels of free tryptophan in the leaves, phloem and xylem saps of Ricinus communis L. were determined by colorimetric assay. Values of 0.38 g ml^-1 in root pressure sap and 96.0 g ml^-1 in phloem sap were recorded. Tryptophan levels were highest in mature and senescing leaves. Levels of indoleacetic acid (IAA) in the phloem sap and leaves were determined by gas chromatography—mass spectrometry using a deuterated internal standard. A mean value of 13.0 ng ml^-1 was recorded in phloem sap. The distribution in the leaves showed an inverse relationship to that of tryptophan, being highest in young leaves.Abbreviations IAA indoleacetic acid - GC-MS Gas chromatography-mass spectrometry - PFP-derivative pentafluoropropionyl-derivative - TLC thin layer chromatography     

Cytokinins and Senescence in Lemon Leaves

The effect of leaf age on cytokinin level was studied in Citrus limon (L.) Burm. f. cv. Eureca. It was found that mature 1-year-old leaves and senescing 2-year-old leaves contained considerably more cytokinins than young 3-month-old leaves. No consistent distinct decrease in cytokinin activity was revealed when the leaf passed from the mature to the senescing state. It was concluded that the results cannot be regarded as supporting the hypothesis that leaf senescence is brought about, at least partially, by a decrease in the level of its cytokinins.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Age-dependent changes in the functions and compositions of photosynthetic complexes in the thylakoid membranes of Arabidopsis thaliana

Photosynthetic complexes in the thylakoid membrane of plant leaves primarily function as energy-harvesting machinery during the growth period. However, leaves undergo developmental and functional transitions along aging and, at the senescence stage, these complexes become major sources for nutrients to be remobilized to other organs such as developing seeds. Here, we investigated age-dependent changes in the functions and compositions of photosynthetic complexes during natural leaf senescence in Arabidopsis thaliana. We found that Chl a/b ratios decreased during the natural leaf senescence along with decrease of the total chlorophyll content. The photosynthetic parameters measured by the chlorophyll fluorescence, photochemical efficiency (F _v/F _m) of photosystem II, non-photochemical quenching, and the electron transfer rate, showed a differential decline in the senescing part of the leaves. The CO₂ assimilation rate and the activity of PSI activity measured from whole senescing leaves remained relatively intact until 28 days of leaf age but declined sharply thereafter. Examination of the behaviors of the individual components in the photosynthetic complex showed that the components on the whole are decreased, but again showed differential decline during leaf senescence. Notably, D1, a PSII reaction center protein, was almost not present but PsaA/B, a PSI reaction center protein is still remained at the senescence stage. Taken together, our results indicate that the compositions and structures of the photosynthetic complexes are differentially utilized at different stages of leaf, but the most dramatic change was observed at the senescence stage, possibly to comply with the physiological states of the senescence process.     

Tissue Distribution of Glutamate Synthase and Glutamine Synthetase in Rice Leaves : Occurrence of NADH-Dependent Glutamate Synthase Protein and Activity in the Unexpanded, Nongreen Leaf Blades

To further explore the function of NADH-dependent glutamate synthase (GOGAT), the tissue distribution of NADH-GOGAT protein and activity was investigated in rice (Oryza sativa L.) leaves. The distributions of ferredoxin (Fd)-dependent GOGAT, plastidic glutamine synthetase, and cytosolic glutamine synthetase proteins were also determined in the same tissues. High levels of NADH-GOGAT protein (33.1 μg protein/g fresh weight) and activity were detected in the 10th leaf blade before emergence. The unexpanded, nongreen portion of the 9th leaf blade contained more than 50% of the NADH-GOGAT protein and activity per gram fresh weight when compared with the 10th leaf. The expanding, green portion of the 9th leaf blade outside of the sheath contained a slightly lower abundance of NADH-GOGAT protein than the nongreen portion of the 9th blade on a fresh weight basis. The fully expanded leaf blades at positions lower than the 9th leaf had decreased NADH-GOGAT levels as a function of increasing age, and the oldest, 5th blade contained only 4% of the NADH-GOGAT protein compared with the youngest 10th leaf blade. Fd-GOGAT protein, on the other hand, was the major form of GOGAT in the green tissues, and the highest amount of Fd-GOGAT protein (111 μg protein/g fresh weight) was detected in the 7th leaf blade. In the nongreen 10th leaf blade, the content of Fd-GOGAT protein was approximately 7% of that found in the 7th leaf blade. In addition, the content of NADH-GOGAT protein in the 10th leaf blade was about 4 times higher than that of Fd-GOGAT protein. The content of plastidic glutamine synthetase polypeptide was also the highest in the 7th leaf blade (429 μg/g fresh weight) and lowest in nongreen blades and sheaths. On the other hand, the relative abundance of the cytosolic glutamine synthetase polypeptide was the highest in the oldest leaf blade, decreasing to 10 to 20% of that value in young, nongreen leaves. These results suggest that NADH-GOGAT is important for the synthesis of glutamate from the glutamine that is transported from senescing source tissues through the phloem in the nongreen sink tissues in rice leaves.     

Quantitative relationship between indole-3-acetic acid and abscisic acid during leaf growth in Coleus blumei

Quantitative determinations by gas chromatography-mass spectrometry ofindole-3-acetic acid (IAA) and abscisic acid (ABA) in growing leaves ofColeusblumei plants show parallel declines in leaf concentrations of bothhormones,except in leaf number 3 (about three-fourths of full size) where IAA level wasthe lowest of those measured. Expansion of the most recently unfurled leaf tofull size serves, in effect, to dilute both IAA and ABA about 1.7 to 1.Althoughabsolute levels of leaf IAA varied as much as an order of magnitude from onebatch of plants to another, ABA levels were proportional to the IAA level withan overall correlation coefficient of 0.91. Evidence, both correlative andcausal, for the determination of ABA status by IAA—and of IAA status byABA—in leaves and other developing organs is summarized.     

Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of ³⁵SO₄²⁻ to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33-fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 μm, while for adenosine phosphosulfate, the Km was 1.35 μm. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3′-phosphoadenosine-5′-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots.     

Environmental control of canopy dynamics and photosynthetic rate in the evergreen tussock grass Stipa tenacissima

Seasonal changes in leaf demography and gas exchange physiology in the tall evergreen tussock grass Stipa tenacissima, one of the few dominant plant species in the driest vegetation of Europe, were monitored over a period of two years at a field site in semi-arid south-eastern Spain. Three age-classes of leaves – young, mature and senescent – were distinguished in the green canopy. Production of new leaves and extension growth of older leaves occurred exclusively from October–November to May–June. The rate of extension was significantly correlated with gravimetric soil water content. Leaf growth ceased after gravimetric soil water content fell below 0.015 g g^–1 at the beginning of the dry season which corresponded to pre-dawn leaf water potentials of -3.0 MPa. Leaf senescence and desiccation reduced green leaf area by 43–49% during the dry season. Diurnal changes in the net photosynthetic rate of all three cohorts of leaves were bimodal with an early morning maximum, a pronounced midday depression and a small recovery late in the afternoon. Maximum photosynthetic rates of 10–16 mol CO₂ m^–2 s^–1 were attained from November 1993 to early May 1994 in young and mature leaves. Photosynthetic rate declined strongly during the dry season and was at or below compensation in September 1994. Gas exchange variables of young and mature leaves were not significantly different, but photosynthetic rate and diffusive conductance to water vapour of senescing leaves were significantly lower than in the two younger cohorts. Leaf nitrogen content of mature leaves varied seasonally between 2.9 and 5.2 g m^–2 (based on projected area of folded leaves), but was poorly correlated with maxima of the photosynthetic rate. There was a stronger linear relationship between the daily maxima of leaf conductance and pre-dawn leaf water potential than with atmospheric water vapour saturation deficit. Seasonal and between-year variation in daily carbon assimilation were caused mainly by differences in climatic conditions and canopy size whereas the effect of age structure of canopies was negligible. Since water is the most important limiting factor for growth and reproduction of S. tenacissima, any future rise in mean temperature, which might increase evapotranspiration, or decrease in rainfall, may considerably reduce the productivity of the grasslands, particularly at the drier end of their geographical distribution.     

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using ¹³[C₆]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 ± 6.5 versus 166.6 ± 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 ± 4.4 versus 263.7 ± 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [¹⁴C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1° and 3° explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.     

Developmental and seasonal changes of stress responsiveness in beech leaves (Fagus sylvatica L.)

The development of beech leaves (Fagus sylvatica L.) was characterized by determination of the pigment and electrolyte concentrations as well as the accumulation of dry mass and specific leaf mass from bud break to senescence. To test the hypothesis that stress tolerance and responsiveness of defences show developmental and/or seasonal changes, leaf discs were either incubated in the absence (control) or presence of paraquat to induce oxidative stress. Controls displayed developmental changes in stress susceptibility ranging from less than 15% of maximum electrolyte leakage in mature leaves to more than 20% leakage in senescent and 36–46% in immature leaves. Paraquat concentrations were chosen to result in about 95% of maximum electrolyte conductivity within 24 h in all developmental stages. Paraquat accumulation was about two‐fold lower in senescent as compared with immature leaves, whereas stress susceptibility, as characterized by the kinetics of the increase in relative leakage, was similar in these developmental stages with 50% of maximum electrolyte conductivity (EC₅₀) = 6·5 h in immature and 7·5 h in senescent leaves. In mature leaves with intermediate paraquat accumulation rates, two classes of stress‐sensitivity were distinguished, namely stress‐resistant and stress‐susceptible leaves with EC₅₀= 9·5 and 5·2 h, respectively. Stress‐resistance of mature leaves was accompanied by a rapid, approximately two‐fold induction of superoxide dismutase activity. Stress‐sensitive mature leaves initially contained high superoxide dismutase activities but showed a rapid, more than six‐ fold loss in activity in 24 h. Correlation of meteorological data with leakage rates suggested that high air temperatures and low precipitation might have been predisposing for loss of resistance against oxidative stress in beech leaves.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Maintenance of High Photosynthetic Rates during the Accumulation of High Leaf Starch Levels in Sunflower and Soybean

Sunflower (cv. “Mammoth Greystripe”) and soybean (Merr. cv. “Amsoy 71”) leaves were exposed to continuous light for at least 52 hours in an attempt to determine the relationship between leaf starch levels and photosynthetic rates. Immature rapidly expanding and relatively mature slowly expanding sunflower leaves were studied. After 52 hours continuous light, the rapidly expanding leaves accumulated high starch levels (3.3 milligrams per square centimeter, 43% of dry weight) with only about a 10% decline from the initial photosynthetic rate of 42 milligrams CO₂ per square decimeter per hour. Under the same conditions, the slowly expanding leaves accumulated less starch, but the photosynthetic rate declined 30%. Soybean leaves, which were slowly expanding, accumulated less starch than sunflower leaves (2.1 milligrams per square centimeter, 34% of dry weight), and their photosynthetic rates declined only about 10% after 54 hours continuous light.