Microdissection and Biochemical Analysis of Plant Tissues

Hampp, Rüdiger; Rieger, A.; Outlaw, William H.

doi:10.1007/978-3-642-83611-4_5

Cited by 21 publications

(14 citation statements)

References 14 publications

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“…Effects of changing environmental factors such as irradiance and temperature on parameters of carbohydrate metabolism were minimized by reducing the time of sampling (1100-1300 hours). Homogenization and lyophilization of individual leaves were as described by Hampp, Rieger & Outlaw (1990). Chlorophyll was extracted in 95 % A7',A?^-dimethylformamide and determined photometrically according to Inskeep & Bloom (1985).…”

Section: Methodsmentioning

confidence: 99%

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

et al. 1997

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SUMMARYBirch cuttings {Betula pendula Roth) were grown in a sand-culture system with two concentrations (0-05, HF and 0-005%, LF) of fertilizer containing macronutrients and micronutrients, and were exposed to 90/40 nll"^ O3 (day/night) and < 3 nl T^ O3 (control) for one growing season in the field fumigation chambers at Birmensdorf (Switzerland). Leaves of different ages were analysed for gas exchange, contents of chlorophyll, protein, and for metabolites as well as enzyme activities of carbohydrate metabolism.Ozone reduced net photosynthesis and chlorophyll contents in mature leaves of both fertilization treatments, whereas that of protein was only reduced in high-fertilized plants (HF). However, net photosynthesis, chlorophyll, and protein increased in young leaves of low'-fertilized plants (LF). The effects of ozone on enzyme activities of carbohydrate metabolism were most pronounced in leaves of LF plants. Specific activities of the sucrose-cleaving enzymes, sucrose synthase and alkaline invertase, were induced, whereas acid invertase was unchanged. Extractable activity of sucrose phosphate synthase, which is a key enzyme of sucrose synthesis, was reduced. Levels of fructose 2,6-bisphosphate, an inhibitor of sucrose synthesis, were increased in leaves of O3/LF plants, but reduced in O3/HF plants. In addition, activities of enzymes involved in starch metabolism, ADP-glucose pyrophosphorylase and starch phosphorylase, were lowered in ozone-treated samples and the ratio of ATP: ADP was increased.It is concluded that chronic ozone exposure leads to an inhibition of sucrose synthesis and favours sucrose degradation. This effect is modulated by the nutrient status of the plants, indicating higher O3 tolerance in HF plants. Furthermore, as the metabolic responses in the ozone-treated samples resemble very closely those observed under end-product inhibition of photosynthesis, we assume that the O3 effect is mainly due to reduced photosynthate export.

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Section: Methodsmentioning

confidence: 99%

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

et al. 1997

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“…2 and 3. Subsampling was done by microdissection (for further details see Lowry and Passonneau 1972;Hampp et al 1990;Rieger and Hampp 1991).…”

Section: Methodsmentioning

confidence: 99%

Compartmentation of soluble carbohydrates, of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves

Rieger

Lutz

Hampp

1992

Planta

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Quantitative histochemistry was used to investigate the tissue-specific compartmentation of soluble carbohydrates (sucrose, glucose, fructose), starch and malate in the laminar pulvinus, leaf blade and petiole of Phaselous coccineus L. at day and night positions of diurnal leaf movement. Total carbohydrate levels measured in a series of cross sections along individual pulvini of 24-d-old plants showed only small differences between the day and night positions of the respective leaf. In contrast, the level of malate changed during diurnal leaf movement, especially in the central part of a pulvinus. The levels of glucose and fructose in the pulvinus increased towards the transition zones between the pulvinus and lamina, and pulvinus and petiole, and this trend was even more pronounced for starch. By contrast, sucrose levels were highest in the pulvinus proper. The transverse compartmentation of metabolites was studied in distinct, approx. 0.5-mm-thick tissue slices from the central part of a pulvinus. These were dissected further into up to 14 distinct subsamples (bundle, bundle sheath, motor tissues, flanks). Irrespective of the position of the leaf (day or night), the central vascular core and the surrounding bundle sheath had high levels of sucrose (up to 500 mmol-(kg DW)(-1)) and low levels of glucose and fructose (below 100 mmol-(kg DW)(-1)), while in the cortex the situation was reversed. In the night position the level of sucrose decreased by approx. 30% in the bundle sheath and the central vascular core but not in the other sections. We thus suggest that because of the relatively small diurnal changes in their cortical pools, soluble sugars are not involved in the osmotic processes resulting in leaf movement. In contrast, pulvini from 14-d-old plants showed an interesting diurnal change in starch and malate pools in the outermost layer of the extensor. Here starch increased at night while the malate pool was lowered nearly stoichiometrically. Inverse pool sizes were found in the day position of the respective leaves. Although less significant, the opposite diurnal variation occurred in samples taken from the flexor region. We thus were able to locate areas of different carbohydrate activities in the laminar pulvinus of P. coccineus. The central vascular core, including the bundle sheath, is involved in temporary storage of photoassimilates, and the cortical regions are responsible for osmotically driven leaf movement. The results are discussed with respect to guard-cell physiology.

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“…3. Their dry weights were determined by means of an ultra-microbalance (Ultramicro 4508 MP 8; Sartorius, Grttingen, FRG) or with a quartz-fiber balance (sample size below 200 ng; Lowry and Passonneau 1972; see also Hampp et al 1990). The respective cut-outs of 3-4 discs (as shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

Intercellular compartmentation of basic carbon pathways in motor organs (pulvini) of leaves of Phaseolus coccineus L.

Rieger

Hampp

1991

Planta

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The activities of enzymes which catalyze one step in each of the five major carbon pathways in green plants were measured in secondary pulvini and other tissues of Phaseolus coccineus L. leaves. We were able to detect activities of fumarase (EC 4.2.1.2; tricarboxylic-acid pathway), NAD-glyceraldehyde-phosphate dehydrogenase (NAD-GAPDH, EC 1.2.1.12; glycolysis), 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44; oxidative pentose-phosphate pathway), ribulose-1, 5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39; photosynthetic carbon-reduction pathway), and of hydroxypyruvate reductase (HP-R, EC 1.1.1.81; photosynthetic carbon-oxidation pathway). On a protein basis the activities of Rubisco and HP-R in pulvinar regions were very low (below 1 and 2 mol · (kg protein) (--1) · h(--1), respectively), but the activities of fumarase and NAD-GAPDH were between 10- and 5-fold higher compared with the laminar tissue (up to 7 and 50 mol · (kg protein)(--1) · h(--1), respectively). Similarly, the protein specific activities of 6-PGDH were increased in the pulvinus (3-4 compared with approx. 1 mol · (kg protein)(--1) · h(--1) in the leaf blade). No differences in specific activities were detected between day and night positions of the leaves. By applying quantitative histochemical techniques we determined the longitudinal and transversal compartmentation of the activities of fumarase, NAD-GAPDH, and 6-PGDH in pulvinar tissues. Levels of activity of all three enzymes increased towards the middle part of the pulvinus. Here, expressed on a dry-weight (DW) basis, the analysis of cross sections showed highest activities in the outer parts of the extensor in the order given, approx. 0.6, 5, and 0.25 mol · (kg DW)(--1) · h(--1) for fumarase, NAD-GAPDH and 6-PGDH. When related to protein, levels of activity were comparably high within the inner parts of extensor and flexor, and partly also in the abaxial part of the bundle (fumarase, 6-PGDH). The tissue-specific compartmentation of the respective activities is discussed in relation to leaf movement and shows parallels with guard-cell function.

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Microdissection and Biochemical Analysis of Plant Tissues

Cited by 21 publications

References 14 publications

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

Ozone‐induced accumulation of carbohydrates changes enzyme activities of carbohydrate metabolism in birch leaves

Compartmentation of soluble carbohydrates, of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves

Intercellular compartmentation of basic carbon pathways in motor organs (pulvini) of leaves of Phaseolus coccineus L.

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