Quantitative enzyme cytochemistry in plant biotechnology

Gahan, P. B.

doi:10.1002/pca.2800020302

Cited by 11 publications

(9 citation statements)

References 54 publications

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“…The use of NBT staining to visualize enzyme activity has been described before. 7,10 In the present study, NBT staining was used to visualize the localization of sucrose synthase, invertase, hexokinase, UGPase and AGPase in growing rice grains (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

Jeng¹,

Tseng²,

Cs³

et al. 2005

J Sci Food Agric

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An in situ nitro-blue tetrazolium (NBT) staining method was used to locate the activities of enzymes involved in starch synthesis in developing grains of field grown rice (Oryza sativa L.) cultivar Tainung 67 and its early ripening mutant SA 419. The results indicated that all the tested enzymes, including sucrose synthase, invertase, hexokinase, ADP-glucose pyrophosphorylase, UDPglucose pyrophosphorylase and starch synthase, were detectable in developing rice endosperms by using NBT staining. The activity of these enzymes was also measurable by using chemical assays. The expression of enzyme activity, as indicated by the formation of blue formazan precipitate, in growing grain of Tainung 67 were visualized in entire endosperm at 11 days after anthesis (DAA). However, NBT staining shifted from central to peripheral region of endosperm after 18 DAA, and the staining disappeared at 25 DAA. Similar staining patterns were also observed in the growing grains of mutant SA 419, but the shift of staining from central to peripheral endosperm occurred at 11 DAA. Electron microscopy examinations showed that the growing patterns of starch granules, sampled from central and peripheral regions of endosperm, varied between two cultivars, with SA 419 growing faster than Tainung 67. Both cultivars showed that the shifts of NBT staining pattern coincided with the changes in the growing pattern of starch granules located in central and peripheral regions of endosperms. The NBT staining results showed that the activities of sucrose to starch conversion enzymes in mutant SA 419 declined and disappeared earlier than its wild-type Tainung 67 and therefore it ripened earlier than cultivar Tainung 67. (c) 2005 Society of Chemical Industry

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Section: Discussionmentioning

confidence: 99%

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

Jeng¹,

Tseng²,

Cs³

et al. 2005

J Sci Food Agric

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“…12 The rate of activity was derived from the slope of the linear regression line obtained by plotting absolute integrated absorbance per mm 3 of reacted cytoplasm versus time of incubation. 11 In addition to measuring the rates of G6PD activity in the shoot apices, leaf primordia (1 and 2) were also monitored in some apices.…”

Section: Quantitative Enzyme Cytochemistrymentioning

confidence: 99%

Calcium stimulation of glucose-6-phosphate dehydrogenase activity in shoot apices ofSpinacia oleracea during floral evocation

Gahan

Ishkhanes

Crèvecœur

et al. 1998

Cell Biochem. Funct.

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The earliest biochemical marker of¯oral evocation in the shoot apex of S. oleracea is the doubling of the rate of glucose-6-phosphate dehydrogenase (G6PD) activity 12±15 h after transfer of 4-week-old plants from short days to continuous light i.e. 1±2 h after the leaves are raised to the¯oral state. Quantitative cytochemical analysis of G6PD activity in the vegetative apices showed that addition of 10 À 7 M Ca 2 to the cytochemical enzyme reaction medium for G6PD activity raises the rate of enzyme activity to that seen in the induced apices. Higher concentrations of Ca 2 result in G6PD inhibition in the vegetative apices and any added Ca 2 at concentrations of 10 À 7 M or higher inhibit the G6PD activity seen in both the induced apices and leaf primordia of both types of apex. The addition of EGTA abolishes the cytochemical reaction. The ability of the Ca 2 to activate the G6PD activity in addition to the incubation medium occurs during the periods of 8±11 h of continuous light, but is already lost by 12 h when no change is achieved by Ca 2 treatment. This can be interpreted as indicating a point in time close to the moment of oral evocation. A model is proposed in which Ca 2 is able to activate the inactivated-G6PD molecules in the vegetative apex through increased Ca 2¯u x possibly through the action of plasmalemmal Ca 2 -ATPase activity as part of the¯oral evocation process.

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“…Measurements of enzyme activities in situ can be achieved either by microbiochemical techniques or by quantitative histochemistry (Van Noorden, 1989;Gahan, 1991;Van Noorden & Butcher, 1991). This second approach uses tissue sections on which enzyme reactions can be performed and the rate of the enzyme activity can be determined either by end-point or continuous monitoring measurements.…”

Section: Introductionmentioning

confidence: 99%

“…This second approach uses tissue sections on which enzyme reactions can be performed and the rate of the enzyme activity can be determined either by end-point or continuous monitoring measurements. In the past, the most commonly used systems for quantitative enzyme histochemistry in animal (Butcher, 1972;Altman, 1975;Van Noorden & Butcher, 1991) as well as in plant tissues (Gahan, 1991) have been scanning and integrating cytophotometers. It is now possible to collect equivalent data more quickly and more easily with image analysers.…”

Section: Introductionmentioning

confidence: 99%

“…However, to our knowledge, this approach does not yet appear to have been exploited in plant histochemistry. Quantitative enzyme histochemistry in 0018-2214 9 1996 Chapman & Hall plants by microdensitometry has been used to study specific problems, such as initiation of carboxylase activity by hormones or involvement of glucose-6-phosphate dehydrogenase (G6PDH) activity in dormancy break or flowering induction (Gahan, 1991). It appears to be a method of choice to obtain information on metabolic activities in small samples of tissues, such as the shoot apical meristem (SAM), which are difficult to study by biochemical techniques.…”

Section: Introductionmentioning

confidence: 99%

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Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

et al. 1996

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A relatively low-cost computer-assisted image analysis system is described. Software has been specifically written for the continuous monitoring of absorbance readings on cryostat sections of plant tissues incubated in media to reveal enzyme activities. The equipment was tested by quantifying glucose-6-phosphate dehydrogenase activity in cryostat sections from shoot apices of spinach plants. The reaction rate of the dehydrogenase activity was monitored at two incubation temperatures, 20 degrees C and 30 degrees C. Control incubations were performed in media lacking substrate. The specific test minus control reaction at 30 degrees C was twice that at 20 degrees C. Variation of the substrate concentration at 30 degrees C yielded a Km value of 0.37 mM. These preliminary results show that our image analysis system can be used for kinetic measurements of dehydrogenase activity in frozen tissue sections and constitute a new approach for enzyme histochemistry in the shoot apical meristem.

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Quantitative enzyme cytochemistry in plant biotechnology

Cited by 11 publications

References 54 publications

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

Calcium stimulation of glucose-6-phosphate dehydrogenase activity in shoot apices ofSpinacia oleracea during floral evocation

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

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