Ploidy Effects in Isogenic Populations of Alfalfa (<i>Medicago sativa</i> L.)

Meyers, Steven P.; Brinegar, A. Chris; Schrader, L. E.; Jordan, Douglas B.; Ogren, William L.

doi:10.1104/pp.71.4.966

Cited by 4 publications

(1 citation statement)

References 12 publications

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“…They found that RuBP carboxylase from the two ploidies had the same Km(C02), the same isoelectric point, and constituted a similar proportion of the soluble protein in both cases ( 18,24). A study of an isogenic ploidy series in alfalfa also revealed no differences in specific activity, Kin, or carboxylase/ oxygenase ratio (19), nor in the proportion of soluble protein that RuBP carboxylase constituted (20 (33) and in the specific activity by Seemann et aL (27). Seemann and Berry (28) found differences in the specific activities but not the Km(CO2) ofRuBP carboxylase extracted from spinach and soybean, and this agreed with observed differences in photosynthetic rate.…”

Section: Discussionmentioning

confidence: 99%

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

Evans¹,

Seemann²

1984

Plant Physiol.

125

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The in vitro ribulose-1,5-bisphosphate (RuBP) carboxylase activity per unit of leaf nitrogen was found to be 30% greater in Triticum aestiaum than in T. monococcum. This was due to a higher specific activity of the enzyme from T. aestirxm, as the amount of RuBP carboxylase protein per unit of total leaf nitrogen did not differ between the genotypes. The occurrence of higher specific activity of RuBP carboxylase is shown to correlate with possession of the lrge subunit derived from the B genome of wheat.Despite the greater RuBP carboxylase activity per unit of leaf nitrogen in T. aestivum, the initial slopes ofcurves relating rate ofC02 assimilation to intercellular p(CO2) are similar in T. aestivuam and T. mowococcum for the same nitrogen content per unit leaf area. The similarity of the initial slopes is the result of a greater resistance to CO2 transfer between the intercellular spaces and the site of carboxylation in T. astivum than in T. monococcum.Studies ofthe photosynthetic performance ofplants have often been made using a wide variety of species representing diverse evolutionary backgrounds. One advantage of studying the physiology of wheat, apart from the fact that much is already known, is that comparisons can be made between species from different stages in the evolution of the crop. The derivation of modem hexaploid wheat has been well established from a combination of classical genetic and taxonomic work (25) with more recent DNA and protein techniques (5, 30).Several studies have used a broad series of wheats to examine the changes in photosynthetic capacity that have occurred with domestication and the increase in ploidy. Evans and Dunstone ( 11) showed that there has been a parallel increase in the size of both seeds and leaves which is associated with a decline in the maximum rate of photosynthesis per unit of leaf area. This decline was thought to be associated with increased mesophyll resistance due to the lower surface to volume ratio of the larger cells in the hexaploid. However, Dunstone et al. (8) This study examines differences in biochemical and anatomical limitations to photosynthesis between primitive and modem lines of wheat. We have concentrated on in vivo and in vitro analysis of the performance of RuBP3 carboxylase in relation to photosynthetic capacity of the intact leaf. Differences in photosynthesis between wheat genotypes could result from differential nitrogen allocation to RuBP carboxylase protein, alterations in the kinetic properties of the enzyme, or anatomical differences which affect the CO2 pressure at the site of carboxylation. We have examined these possibilities through a comparison of a modem hexaploid wheat, T. aestivum, and a diploid ancestor, T. monococcum. We have also extended this study to include a more thorough examination of the relationship between kinetic properties of RuBP carboxylase and wheat genome.MATERIALS AND METHODS Plant Material. Seed of the wheat accessions (Table I)

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Section: Discussionmentioning

confidence: 99%

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

Evans¹,

Seemann²

1984

Plant Physiol.

125

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Breeding for Improved CO2 Fixation

Buzzell

Buttery

1984

Applications of Genetic Engineering to Crop Improvement

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A Simple and Sensitive DNA Assay for Plant Extracts

Baer

Meyers²,

Molin³

et al. 1982

Plant Physiol.

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extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4',6'-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCI to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.Expressing enzyme activities per cell rather than per unit weight, Chl, or protein is extremely important in some areas of biologic research. The simplest and most accurate method of calculating the cell number in most higher plant tissues is by determining the amount of DNA per cell and per tissue preparation. Most available methods for the quantitation of DNA require considerable manipulation of the samples, which may result in a loss of DNA. Such methods are time-consuming and not very sensitive. The commonly used diphenylamine method (4) suffers from these limitations.The fluorescent dye DAPI3 complexes with DNA to give a product with fluorescence intensity several times greater than the dye alone. Using this property, Kapuscinski and Skoczylas (8) have shown that the specificity and sensitivity of this reaction could be used for a sensitive DNA assay. The method permitted the estimation of concentrations of DNA as low as 0.5 ng/ml, even in the presence of a 20-fold excess of RNA. They showed that the fluorescence intensity is highest when DAPI Extract of Protoplasts. The method for the preparation of alfalfa protoplasts is described elsewhere (11). An aliquot (0.2 ml) of suspension containing approximately one million protoplasts/ ml was centrifuged at 60g for 10 min. The supernatant was discarded, and the pellet was stored at -15°C. The thawed pellet was suspended in 0.3 ml buffer (2.0 M NaCl, 10 mM EDTA, 10 mM Tris [pH 7.0]). Where indicated, sonication was performed for 15 s or 1% (w/v) sarkosyl was added to the suspension buffer.Chloroform Treatment. Chloroform (1.5 volumes) was added to the homogenate and vigorously mixed with a Vortex mixer. The phases were separated by centrifugation at l,000g for 10 min, and the aqueous supernatant was used for fluorimetric measurements.No loss of DNA resulted from chloroform treatment of homogenates or calf thymus DNA solution (data not shown).Fluorimetric Measurements. Fluorescence was measured with a Perkin-Elmer 650-1OS fluorescence spectrophotometer with the excitation and emission wavelengths set at 350 nm and 450 nm, respectively. A stock solution of DAPI (1 mg/100 ml H20) was prepared and could be stored at 4°C for several months. DAPI was used at final c...

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Ploidy Effects in Isogenic Populations of Alfalfa (Medicago sativa L.)

Cited by 4 publications

References 12 publications

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

Breeding for Improved CO2 Fixation

A Simple and Sensitive DNA Assay for Plant Extracts

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