Criteria of Intactness and the Photosynthetic Activity of Spinach Chloroplast Preparations

Lilley, Ross McC.; Fitzgerald, Mabel Purefoy; Rienits, K.G.; Walker, David A.

doi:10.1111/j.1469-8137.1975.tb01365.x

Cited by 416 publications

(149 citation statements)

References 18 publications

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“…As summarized above, the incubating media were also different for the two methods (2,10). Chloroplast intactness was determined by the ratio of the 02 evolution rates of shocked and unshocked chloroplasts using ferricyanide as electron acceptor (8).…”

Section: Methodsmentioning

confidence: 99%

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Anderson

Done²

1978

Plant Physiol.

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Chloroplasts were prepared from peas (P_m sadvum) In glucosephosphate medim. In the presence of DL-glyceraldehyde, they catabzed nitrite t 0s evolution (mean of 13 preparatons, 17.5 Mmole per mg chlorophyi per hour, SD 3.64). The optimum concentration of nirte was 0.5 mM; 0.12 mM nitrite supported V11J2. The reaction was accompaled by the cou t of ntrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. hI short experiments (less than 10 minutes) the 0s to nirte ratio approached 1.5 The enzymes glutamine synthetase and glutamate synthetase, which catalyze reactions now known to be basic for N assimilation, have been located in chloroplasts (7,11,13) and shown to be coupled to light-dependent electron transport. Thus, glutamate synthetase activity can be monitored by (glutamine plus a-ketoglutarate)-dependent 02 evolution (2). In the presence of the cofactors ADP, PPi, and Mg2+, pea chloroplasts also catalyze (ammonia plus a-ketoglutarate)-dependent 02 evolution which we attributed to assimilation of ammonia into glutamate via photosynthetically coupled4 glutamine synthetase and glutamate synthetase (1).Spinach chloroplasts in the light actively reduce nitrite (9,10,14); approximately 60 to 90% of the nitrite-N consumed is incorporated into amino-N (9, 10). Spinach chloroplasts also catalyze nitrite-depentent 02 evolution (10). According to current theory, 'This work was conducted while the authors were on sabbatical leave from their respective universities. nitrate-N is reduced to nitrite in the cytoplasm and further reduced to ammonia by nitrite reductase within the chloroplast. The ammonia so formed is incorporated via glutamine into glutamate in the chloroplast in reactions catalyzed by glutamine synthetase and glutamate synthase. In this event, nitrite-N should compete with glutamine-N and ammonia-N in the latter stages of the N assimilation pathway. We previously reported, however, that nitrite-dependent and (glutamine plus a-ketoglutarate)-dependent 02 evolution activities catalyzed by pea chloroplasts did not compete (2) implying that ammonia produced from nitrite was not assimilated by pea chloroplasts under the prevailing experimental conditions. We give a report in this paper of a study of lightdependent reduction and assimilation ofnitrite by pea chloroplasts under conditions which (a) support and (b) do not support (ammonia plus a-ketoglutarate)-dependent 02 evolution. MATERIALS AND METHODSSeedlings of pea (Pisum sativum cv. Feltham First) were used. The methods for growing peas, measurement of Chl and chloroplast preparation (referred to in this paper as method 1) were as described previously (2). 02 evolution was measured polarographically and calibrated against air-saturated water at 25 C as before (2). Chloroplasts prepared by method I were incubated in sorbitol-HEPES medium (2) and were known to catalyze (glutamine plus a-ketoglutarate)-dependent 02 evolution and, in the presence of ADP, MgCl2 and PPi (ammonia plus a-ketoglutarate)-dependent 02 evolution (1, 2). Some aspect...

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Section: Methodsmentioning

confidence: 99%

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Anderson

Done²

1978

Plant Physiol.

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“…Soluble leaf protein was extracted into 1 mL of ice-cold, N 2 -sparged extraction buffer [50 mM EPPS-NaOH, pH 8, 0.5 mM EDTA, 2 mM DTT, 1% (v/v) plant protease inhibitor cocktail (Sigma-Aldrich), and 1% (w/v) PVPP)] with 5 mM MgCl 2 using 2-mL glass homogenizers (Wheaton; Sharwood et al, 2016). The lysate was centrifuged for 30 s (16,000g, 4°C), and 10 mL of the soluble protein was assayed for initial and total Rubisco activities using an NADH-coupled enzyme method at 25°C (Lilley et al, 1975;Sharwood et al, 2016). To measure initial Rubisco activity, RuBP (0.4 mM) was added to the buffer prior to the addition of extract (10 mL) to start the assay.…”

Section: Leaf Protein Extraction and Rubisco Activation And Content Mmentioning

confidence: 99%

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells

et al. 2017

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In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts. We conclude that BSD2 does not play a vital role in M cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO 2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates.

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“…Resuspended the chloroplast pellets in a smaller volume of the above buffer. Intactness of purified chloroplasts was measured according to ferricyanide reduction test (Lilley et al 1975) using Clark-type O 2 electrode (Hansatech Instruments Ltd., England). The percentage intactness of chloroplasts was 80-85 % up to 21 DAA and 68-74 % at 28 DAA.…”

Section: Methodsmentioning

confidence: 99%

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants

Semwal

Singh

Khanna‐Chopra

2014

Physiol Mol Biol Plants

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Reproductive sinks regulate monocarpic senescence in crop plants. Monocarpic senescence was studied in wheat fertile (cv. HW 2041) and its isonuclear cytoplasmic male sterile (CMS) line. CMS plants exhibited slower rate of senescence accompanied by longer green leaf area duration and slower deceleration in chlorophyll, protein content, P N and rubisco content coupled with lower protease activities than fertile (F) plants. CMS plants also exhibited lower ROS levels and less membrane damage than F plants. CMS plants maintained better antioxidant defense, less oxidative damage in chloroplast and higher transcript levels of both rbcL and rbcS genes during senescence than F plants. F plants exhibited early induction and higher expression of SAGs like serine and cysteine proteases, glutamine synthetases GS1 and GS2, WRKY53 transcription factor and decline in transcript levels of CAT1 and CAT2 genes than CMS plants. Hence, using genetically fertile and its CMS line of wheat it is confirmed that delayed senescence in the absence of reproductive sinks is linked with slower protein oxidation, rubisco degradation and delayed activation of SAGs. Better antioxidant defense in chloroplasts at later stages of senescence was able to mitigate the deleterious effects of ROS in CMS plants. We propose that delayed increase in ROS in cytoplasmic male sterile wheat plants resulted in delayed activation of WRKY53, SAGs and the associated biochemical changes than fertile plants.

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Criteria of Intactness and the Photosynthetic Activity of Spinach Chloroplast Preparations

Cited by 416 publications

References 18 publications

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants

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