Sodium, Potassium, Chloride, and Betaine Concentrations in Isolated Vacuoles from Salt-Grown Atriplex gmelini Leaves

Vacuoles were isolated via protoplasts from the leaves of a halophyte Atriplex gmelini C.A.Mey., grown in culture solution supplemented with 250 millimolar NaCl. Lysis of the protoplasts was induced by lowering the medium osmolarity (1.2 to 1.0 molar sorbitol) and adding a detergent, a synthesized cholate derivative, 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate at a concentration of 0.5 millimolar and the resulting vacuoles were purified by successive dilution and floatation. Isolated vacuoles contained almost the same concentration of sodium (569 millimolar) and chloride (260 millimolar) as recorded in protoplasts (582 and 254 millimolar, respectively), suggesting that the vacuoles are the major sequestration site of NaCl in leaves of halophytes. Betaine concentration in the protoplasts was about 16 millimolar, while that in vacuoles was only about 0.24 millimolar, indicating that betaine is accumulated in the cytoplasm as a compatible solute.     

Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum)

K⁺ content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCl₂ solution (1°C). From time-course curves of cumulative K⁺ diffusion from the tissue, the amount of K⁺ of extracellular origin was estimated. Apoplastic K⁺ contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K⁺ were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K⁺ content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K⁺ concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb⁺ label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K⁺ content was found to be highly sensitive to changes in xylem solution concentration.     

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4°C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl₂, 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58°C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25°C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Stromal Phosphate Concentration Is Low during Feedback Limited Photosynthesis

It has been hypothesized that photosynthesis can be feedback limited when the phosphate concentration cannot be both low enough to allow starch and sucrose synthesis at the required rate and high enough for ATP synthesis at the required rate. We have measured the concentration of phosphate in the stroma and cytosol of leaves held under feedback conditions. We used non-aqueous fractionation techniques with freeze-clamped leaves of Phaseolus vulgaris plants grown on reduced phosphate nutrition. Feedback was induced by holding leaves in low O₂ or high CO₂ partial pressure. We found 7 millimolar phosphate in the stroma of leaves in normal oxygen but just 2.7 millimolar phosphate in leaves held in low oxygen. Because 1 to 2 millimolar phosphate in the stroma may be metabolically inactive, we estimate that in low oxygen, the metabolically active pool of phosphate is between negligible and 1.7 millimolar. We conclude that halfway between these extremes, 0.85 millimolar is a good estimate of the phosphate concentration in the stroma of feedback-limited leaves and that the true concentration could be even lower. The stromal phosphate concentration was also low when leaves were held in high CO₂, which also induces feedback-limited photosynthesis, indicating that the effect is related to feedback limitation, not to low oxygen per se. We conclude that the concentration of phosphate in the stroma is usually in excess and that it is sequestered to regulate photosynthesis, especially starch synthesis. The capacity for this regulation is limited by the coupling factor requirement for phosphate.     

Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

Photosynthesis and photosynthate partitioning in n(2)-fixing soybeans

Leaf area, chlorophyll content, net CO₂ photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. A second set (Nod+/−) was inoculated and had N₂ fixation as its sole source of nitrogen. A third group (Nod⁻) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. The mean net micromole CO₂ uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/− test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/− utilized their chlorophyll more efficiently for photosynthetic CO₂ uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/− but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/− sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation.     

Termination of nutrient import and development of vein loading capacity in albino tobacco leaves

The sink-source conversion in developing leaves of tobacco (Nicotiana tabacum L.) was studied to determine whether import termination is caused by the onset of export or is related to achievement of positive carbon balance. Albino shoots were grown in vitro and grafted to detopped stems of green tobacco plants. Termination of import was studied by providing mature leaves of the stock plant with ¹⁴CO₂ and detecting the presence of labeled nutrient in developing albino leaves by whole-leaf autoradiography. In albino leaves, import terminated progressively in the basipetal direction at the same stage of development as in leaves of green shoots. Starch was not present in the plastids of mesophyll cells of mature albino leaves but starch was synthesized when discs were cut from these leaves and incubated on 3 millimolar sucrose. Import ceased progressively in developing green leaves even when photosynthesis was prevented by darkening. It was concluded that cessation of import does not require achievement of positive carbon balance and is not the direct result of export initiation.

To determine whether vein loading capacity develops in albino leaves, discs were cut from mature leaves and floated on [¹⁴C]sucrose solution. Uptake of label into the veins was detected by autoradiography and this uptake was sensitive to the phloem loading inhibitor p-chloromercuribenzenesulfonic acid. However, the amount of label taken up by veins in albino leaves was less than that taken up by veins of mature green leaves.

     

Ion Homeostasis in Chloroplasts under Salinity and Mineral Deficiency: II. Solute Distribution between Chloroplasts and Extrachloroplastic Space under Excess or Deficiency of Sulfate, Phosphate, or Magnesium

Spinach (Spinacia oleracea var “Yates”) plants grown hydroponically were exposed to an excess or deficiency of various mineral ions. Solutes were measured in leaf extracts and in isolated intact chloroplasts. Under phosphate (120 millimoles per liter NaH₂ PO₄), sulfate (200 millimolar per liter (Na₂ SO₄), or magnesium excess (150 millimolar per liter MgCl₂), concentrations of these ions in leaf extracts increased, but in chloroplasts, concentrations of all ions remained constant. Concentrations of quarternary ammonium compounds in chloroplasts increased. Under mild phosphate or magnesium deficiency, concentrations of these ions decreased in chloroplasts less than in whole leaf extracts. Under severe sulfate deficiency causing chlorosis in younger leaves, sulfate concentrations in chloroplasts remained even unchanged, despite a drastic decrease of sulfate concentrations both in green and in chlorotic leaves. Together with results from a companion study (G Schröppel-Meier, WM Kaiser 1988 Plant Physiol 87: 822-827) our data demonstrate that leaf cells are able to keep the concentrations of several mineral ions rather constant in metabolically active compartments even at extremely large variations of ion concentrations in the culture solution and in the leaves.     

Adenine Nucleotide Levels, the Redox State of the NADP System, and Assimilatory Force in Nonaqueously Purified Mesophyll Chloroplasts from Maize Leaves under Different Light Intensities

Recently, a nonaqueous fractionation method of obtaining highly purified mesophyll chloroplasts from maize leaves was established. This method is now used to determine adenine nucleotide levels, the redox states of the NADP system, Pi levels and dihydroxyacetone phosphate/3-phosphoglycerate ratios in mesophyll chloroplasts of Zea mays L. leaves under different light intensities. The sum of the ATP, ADP, and AMP levels was estimated to be 1.4 millimolar and the ATP/ADP ratio was 1 in the dark and 2.5 to 4 in the light. The adenine nucleotides were equilibrated by adenylate kinase. The total concentration of NADP(H) in the chloroplasts was 0.3 millimolar in the dark and 0.48 millimolar in the light. The ratio of NADPH/NADP was 0.1 to 0.18 in the dark and 0.23 to 0.48 in the light. The Pi level was estimated to be 20 millimolar in the dark and 10 to 17 millimolar in the light. The 3-phosphoglycerate reducing system was under thermodynamic equilibrium in the light. The calculated assimilatory forces were 8 per molar and 40 to 170 per molar in the dark and the light, respectively. There was no relationship between the degree of activation of pyruvate, Pi dikinase, and adenylate energy charge, or ATP/ADP ratio or ADP level under various light intensities. Only a weak relationship was found between the degree of activation of NADP-malate dehydrogenase and the NADPH/NADP ratio or NADP(H) level with increasing light intensity. A possible regulatory mechanism which is responsible for the regulation of activation of pyruvate,Pi dikinase and NADP-malate dehydrogenase is discussed.     

Effects of ethephon on aging and photosynthetic activity in isolated chloroplasts

Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 25°C with 2-chloroethylphosphonic acid (ethephon) under light (0.16 milliwatts per square centimeter) and dark conditions. Ethephon at 1 micromolar (0.1445 ppm), 0.1 and 1 millimolar, or 5 microliters ethylene promoted the deterioration of chloroplasts, increased proteolysis, and reduced the chlorophyll content and PSI and PSII during 72 hours under both light and dark conditions. The decline in PSI and PSII occurred prior to a measurable loss of chlorophyll. The loss of photosynthetic activity affected by ethephon was initiated prior to 12 hours of incubation. After 24 hours in light, 0.1 millimolar (1.445 ppm) epthephon significantly reduced PSI and PSII and promoted the total free amino acid liberation in isolated chloroplasts. In darkness the rate of loss of PSI activity was about 50% of that in light. After 24 hours, in light at 1 millimolar epthephon, PSII activity was 55% of the control, yet nearly 90% of the chlorophyll remained, which indicates that the loss of thylakoid integrity was promoted by ethephon. Ethylene injected in the chloroplast medium at 5 microliters (0.22 micromolar per milliliter) reduced PSI by nearly 50% of the initial in 12 hours. In leaf sections floated in 5 microliters per milliliter suspension medium, a 36% loss of chlorophyll of the control in 36 hours was observed. Cycloheximide at 0.5 millimolar masked the effect of 1 millimolar ethephon and maintained the initial chlorophyll content during the 72 hour period.     

杜仲叶片和叶柄愈伤组织的诱导和植株再生

本实验以5~6年生杜仲叶片及叶柄为外植体,研究了杜仲愈伤组织诱导及植株再生的方法。结果表明:接种于补加NAA(2.0~4.0 mg/L)或BA(1.0 mg/L)+NAA(2.0~4.0mg/L)的MS培养基上的叶片和叶柄,经21~28d培养后,脱分化形成绿色或浅绿色致密愈伤组织,频率达到70%以上。绿色致密愈伤组织在补加BA(2.25~2.75 mg/L)+NAA(0.15 mg/L)的MS培养基上经过1~2次继代之后,即出现茎芽分化,频率在15%以上,只是其中许多都是畸形苗,正常苗频率较低。此问题尚在研究之中。选择生长健壮的再生植株,切除其基部愈伤组织,然后将切口浸泡在250mg/L无菌ABT生根粉溶液中3~5sec,再插入1/4强度无激素MS培养基中, 2~3周后,在苗基部长出1~3条白色粗壮的不定根,生根频率在60%以上。     

Use of Phloem exudate technique in the study of amino Acid transport in pea plants

The phloem exudation technique using ethylenediaminetetraacetic acid (EDTA) was evaluated in studies of amino acid translocation in Pisum sativum L. seedlings. Exudation of phloem sap from cut petioles of fully expanded leaves was enhanced by EDTA (20 millimolar disodium salt [pH 7.0]). Amino acids (mainly asparagine, homoserine, glutamate, and also aspartate and serine) were present in petiole exudates from EDTA-treated leaves at levels which were commonly 5- to 10-fold (or more) higher compared with water-treated controls. Exudation was greater from darkened leaves, and the pattern of amino acids was markedly different from the more uniform mixture leaking from water-treated controls.     

Nitrate and Carbohydrate Effects on Nodulation and Nitrogen Fixation (Acetylene Reduction) Activity of Lentil (Lens esculenta Moench)

Lentils (Lens esculenta Moench, cv. Tekoas) grown in a nutrient solution containing 15 millimolar nitrate had 84% fewer nodules than lentils grown in nitrate-free nutrient solution. Nodules from the nitrate-grown plants weighed 71% less than nodules from the nitrate-free plants. Nitrate-grown plants also fixed much less nitrogen (measured by acetylene reduction) than the nitrate-free plants. When lentils were grown in a solution containing 15 millimolar nitrate and 75 millimolar fructose, glucose, or sucrose, however, the nitrogen fixation activity of their nodules was similar to that of nodules from nitrate-free plants. Leaves of lentils grown in the nitrate-sugar solutions had only about 7% as much nitrate reductase activity and accumulated only 10% as much nitrate as leaves from lentils grown in the nitrate solution alone. Roots of lentils grown in the nitrate-sugar solutions had similar nitrate reductase activity but accumulated only 17 to 25% as much nitrate as roots from lentils grown in the nitrate solution. The results indicate that the added sugars alleviated the inhibitory effects of nitrate on symbiotic nitrogen fixation not only by increasing the carbohydrate supply so lentils could support both nitrogen fixation and nitrate reduction but also by inhibiting the accumulation of nitrate and, hence, lowering nitrate reductase activity in the leaves.     

Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP

Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M_r value of glycerate kinase as determined by gel filtration (M_r = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.

Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis.

     

Potassium nutrition and translocation in sugar beet

The effect of increased net foliar K⁺ accumulation on translocation of carbon was studied in sugar beet (Beta vulgaris, L. var. Klein E and US H20) plants. Net accumulation of recently absorbed K⁺ was studied by observing arrival of ⁴²K⁺ per unit area of leaf. Labeled K⁺ was added to give an initial concentration at 2 or 10 millimolar K⁺ in mineral nutrient solution. Because the newly arrived K⁺ constitutes a small part of the total leaf K⁺ in plants raised in 10 millimolar K⁺, export of ⁴²K⁺ by phloem was negligible over the 2- to 3-day period; consequently, accumulation is a measure of arrival in the xylem. In leaves from plants in 2 millimolar K⁺, export by the phloem was estimated to be of the same order as import by the xylem; K⁺ per area was observed to remain at a steady-state level. Increasing the supply of K⁺ to 10 millimolar caused arrival in the xylem to increase 2- to 3-fold; K⁺ per area increased gradually in the mature leaves. Neither net carbon exchange nor translocation of sugar increased in response to a faster rate of arrival of K⁺ over a 6- to 8-hour period. In the absence of short-term effects, it is suggested that K⁺-promoted increase in synthetic metabolism may be the basis of the increased carbon assimilation and translocation in plants supplied with an above-minimal level of K⁺.     

Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence

When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1-phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4-dinitrophenol were found to be inhibitors of CO₂ exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Hormones and young leaves control development of cotyledonary buds in tomato seedlings

Hormonal and plant factors regulating the development of the inhibited cotyledonary buds of Lycopersicon esculentum Mill. cv. `Fireball' seedlings were studied. Excision of the immature plumular leaves of 5- to 20- millimeter length significantly stimulated bud development after 2 to 4 days, but excision of leaves exceeding 20-millimeter length was without effect. Apical application of 20 microliters of 5 millimolar abscisic acid significantly promoted development of the cotyledonary buds after 6 days. A subapical ring of 0.1 millimolar concentration of 2,3,5-triiodobenzoic acid (TIBA) in lanolin significantly promoted cotyledonary bud development after 11 days. Twenty microliters of 0.1 millimolar 6-benzylaminopurine (BAP) applied directly to the cotyledonary bud loci significantly promoted bud development, but 1 micromolar gibberellin A_4/7 was ineffective. Application of 0.1 millimolar BAP in lanolin to the petiole or hypocotyl was ineffective. However, application of 0.1 millimolar TIBA as a ring around the petioles of the cotyledons or 1-centimeter on the hypocotyl below the cotyledons significantly promoted cotyledonary bud development.     

The effect of sodium chloride on solute potential and proline accumulation in soybean leaves

Two cultivars of soybean (Glycine max [L.] Merr.) were grown in solution with up to 100 millimolar NaCl. Leaf solute potential was −1.1 to −1.2 megapascals in both cultivars without NaCl. At 100 millimolar NaCl leaf solute potential was −3.1 to −3.5 megapascals in Bragg and −1.7 megapascals in Ransom. The decrease in solute potential was essentially proportional to the concentration of NaCl. In both salt susceptible Bragg and salt semitolerant Ransom, leaf proline was no more than 0.4 micromole per gram fresh weight at or below 20 millimolar NaCl. At 40 and 60 millimolar NaCl, Bragg leaf proline levels were near 1.2 and 1.9 micromoles per gram fresh weight, respectively. Proline did not exceed 0.5 micromole per gram fresh weight in Ransom even at 100 millimolar NaCl. Proline accumulated in Bragg only after stress was severe enough to induce injury; therefore proline accumulation is not a sensitive indicator of salt stress in soybean plants.