Hydrogen Peroxide Synthesis in Isolated Spinach Chloroplast Lamellae

115

Chenopods synthesize betaine in the chloroplast via a twostep oxidation of choline: choline betaine aldehyde betaine.Our previous experiments with intact chloroplasts, and in vivo 1802 labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCI. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.supports Wyn Jones's hypothesis (28) that betaine accumulation is a biochemical adaptation to salinity, with betaine acting as a major cytoplasmic osmolyte in salt-stressed plants (1 1, 17, 26). In stressed or unstressed plants, betaine is synthesized by a two-step oxidation of choline; both steps occur in the chloroplast in spinach and other chenopods (3):-2H -2H Choline -. betaine aldehyde -* betaine In spinach, the second step is catalyzed by a stromal BADH,4 which is encoded by a single nuclear gene (24,25). The enzyme for the first step is not yet known, although two lines of indirect evidence indicate that it is a monooxygenase (which could produce the hydrate form of betaine aldehyde by introducing an OH group at Cl of choline). First, in vivo labeling studies demonstrate that spinach leaf disks incorporate 180 from 1802 into betaine, and that this occurs at the choline -. betaine aldehyde step (9). Second, experiments with intact chloroplasts show that the choline -+ betaine aldehyde reaction requires photosynthetically generated reducing power as well as 02(23). The choline-oxidizing enzyme from spinach chloroplasts would therefore appear to be unlike the choline dehydrogenases (8,12,21)

Section: C]choline Oxidation By a Stromal Enzyme Dependent Upon Redmentioning

confidence: 99%

Evidence for a Ferredoxin-Dependent Choline Monooxygenase from Spinach Chloroplast Stroma

Brouquisse

Weigel²,

Rhodes³

et al. 1989

115

“…Mature leaves were excised, washed, and blotted and intact plastids were prepared. (23). PS parameters for both intact plastid isolates and lamellae isolates were estimated in the same experimental period (see Table III).…”

mentioning

confidence: 99%

“…Chl was extracted from 10 to 15 mg freeze-dried leaf tissue with 10 ml 80% acetone, and Chl was quantitated as described previously (23).…”

mentioning

confidence: 99%

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Robinson¹

1984

Self Cite

116

ABSTRACIResponses of folar and isolted intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia okracea cv Wisconsin Bloomsdal) plants exposed to a shortened photosynthetic peried (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propated contiuosly on a 12-hour light/12-bour dark cycle, 14 to 15 days were requied, subsequent to a shift from 12 to 7 honrs dayength, for 7-howr plants to begin to grow at rates comparable to those of 12-hour daylegth plants. Became of shorter daily dtions of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour thano in the 12-how plants. The resut was that 7-hour plants established a 1.5-to 2.0-fold higer total PS rate than 12-hour phats.Inact chioroplasts isolted from the baves of7-hour plants (7-h PLD) displayed 1.5-to 2.0-fold higher PS rates than plastids isolated from 12-hoor plants . Plastid lamellae prep from 7-and 12-h PID isolates displayed equivalnt rates of ferredoxin-dependent Al? and NADPH photoformation d ng that eler transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in kaves as well as intact PID isolates, of dark to light transitional inceases in Calvin cycle intermediates, eg., ribulose-1,5-bisphosphate (RuBP) and 3-phospboglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bispbosphate phosphatase, indcated that 7-hour plant leaves displayed higher PS rates (tha 12-how plants), became there was a higher maitude of activity of the Calvin cycle.Alhough both the folar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-howr compared with 12-howr plant folg, the higher 7-hour plt total PS rates incated that maximal suose and starch leels did not mediate any 'feedback' inhibition of PS. The higber 7-hour plant foliar and PLD PS rates resulted in higher gluose-l-P leels as well as a higher ratio of 3-PGA:Pi, both factors of which would enance the activity of chloroplast ADP-glcose pyrophospboryblse, and which were attributed to be casal to the highe starch synthesis rates observed in 7-hour plant foliage and PLD isolates.Leaf photosynthetic rate may be regulated by the requirement for photosynthate, i.e. the 'demand' for photoassimilate, exerted by the growth and development of sink organs in the higher plant (8, 11). Silvius et al. (28)

“…In darkness however, the difference in pH between the thylakoid space and stroma was negligible (28). (c) Chloroplasts are known to contain a number of autocatalytic enzymes (20)(21)(22). Ethylene is known to induce several enzymes including catalase and peroxidase which has been thought to have a stabilizing effect on Chl (21).…”

mentioning

confidence: 99%

Effects of Ethephon on Aging and Photosynthetic Activity in Isolated Chloroplasts

Choe¹,

Whang²

1986

Chloroplasts, isolated from the primary leaves of 7-day-old seedlings, were incubated in vitro at 256C 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 chlorophsts. In darkness the rate of loss of PSI activity was about 50%O 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.To promote maturity or color of apples, blackberries, blueberries, cranberries, pineapple, and tomatoes, foliar sprays of an ethephon solution ranging from 200 to 1000 actual ppm have been the recent field practice (24). Ethylene evolved from ethephon has been known to hasten maturity and the senescence phenomena in plant tissues. Ethylene released from ethephon promotes the loss of Chl (4, 18, 1-9), hydrolysis of polymers (12,14), climacteric rise in respiration-(1, 14), and early maturation or accelerated senescence (1,2,13 MATERIALS AND METHODSChloroplasts were isolated from oat (Avena sativa cv. 'Garry Spring') seedlings which were grown for 7 d in vermiculite at 25°C. Cool white fluorescent tubes provided light for 14 h/d at approximately 0.81 mW cm-2 at the plant level. A bundle of 10 g of apical 5-cm leaves were chilled, cut, surface sterilized, finely minced with a razor blade, and homogenized in a freshly prepared chilled isolation medium A (5). This medium A contained 30 mm Tes, 0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl2, 5 mM sodium ascorbate, and 0.1% BSA fraction V and was adjusted to pH 7.4 with 1 N NaOH. Chloroplasts were aseptically isolated at 0 to 4°C from the homogenate as previously described (6)