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

Abstract

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.

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

“…Freeze-dried sections of 14-d-old pulvini were transferred to liquid-nitrogen-cooled glass vials and homogenised with a cooled microdrill without any liquid (for further details see Rieger and Hampp 1991). The resulting powder (between 10 and 200 ~tg dry weight) was extracted under continuous microdrill use on ice for 30 s after addition of 50 I~l 0.5 N KOH (containing 100mM ethanol; Dekker and Richards 1971;Ettel 1981).…”

“…This arrangement was fixed inside an Erlenmayer flask (100 ml) filled with distilled water in such way that the original in-situ angle between the main axis and petiole (approx. 140 ~ was maintained (Rieger and Hampp 1991). Pulvini from these leaves were harvested 14 or 24 d after sowing ("cut-leaf pulvini": CLP).…”

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

“…The method of sample collection, microdissection and homogenisation is described elsewhere (Rieger and Hampp 1991).…”

“…Photosynthesis is probably not the only source of energy as leaf movement also takes place in continuous darkness (e.g., Satter et al 1979). Recently, we were able to show that motor tissues from P. coccineus generally have evolved high-capacity pathways for dissimilation (Rieger and Hampp 1991). Furthermore, we reported evidence that the activity of ribulose bisphosphate carboxylase in motor tissues is considerably lower than in the mesophyll (see also Vanhinsberg and Horton 1990).…”