Determination of the site of CO2 sensing in poplar: is the area-based N content and anatomy of new leaves determined by their immediate CO2 environment or by the CO2 environment of mature leaves?

Exposure to an elevated CO(2) concentration ([CO(2)]) generally decreases leaf N content per unit area (N(area)) and stomatal density, and increases leaf thickness. Mature leaves can 'sense' elevated [CO(2)] and this regulates stomatal development of expanding leaves (systemic regulation). It is unclear if systemic regulation is involved in determination of leaf thickness and N(area)-traits that are significantly correlated with photosynthetic capacity. A cuvette system was used whereby [CO(2)] around mature leaves was controlled separately from that around expanding leaves. Expanding leaves of poplar (Populus trichocarpa×P. deltoides) seedlings were exposed to elevated [CO(2)] (720 µmol mol(-1)) while the remaining mature leaves inside the cuvette were under ambient [CO(2)] of 360 µmol mol(-1). Reverse treatments were performed. Exposure of newly developing leaves to elevated [CO(2)] increased their thickness, but when mature leaves were exposed to elevated [CO(2)] the increase in thickness of new leaves was less pronounced. The largest response to [CO(2)] was reflected in the palisade tissue thickness (as opposed to the spongy tissue) of new leaves. The N(area) of new leaves was unaffected by the local [CO(2)] where the new leaves developed, but decreased following the exposure of mature leaves to elevated [CO(2)]. The volume fraction of mesophyll cells compared with total leaf and the mesophyll cell density changed in a manner similar to the response of N(area). These results suggest that N(area) is controlled independently of the leaf thickness, and suggest that N(area) is under systemic regulation by [CO(2)] signals from mature leaves that control mesophyll cell division.

Stomatal development in new leaves is related to the stomatal conductance of mature leaves in poplar (Populus trichocarpaxP. deltoides).

In general, stomatal density (SD) decreases when plants are grown at high CO2 concentrations. Recent studies suggest that signals produced from mature leaves regulate the SD of expanding leaves. To determine the underlying driver of these signals in poplar (Populus trichocarpaxP. deltoides) saplings, a cuvette system was used whereby the environment around mature (lower) leaves could be controlled independently of that around developing (upper) leaves. A series of experiments were performed in which the CO2 concentration, vapour pressure deficit (D), and irradiance (Q) around the lower leaves were varied while the (ambient) conditions around the upper leaves were unchanged. The overall objective was to break the nexus between leaf stomatal conductance and transpiration and photosynthesis rates of lower leaves and determine which, if any, of these parameters regulate stomatal development in the upper expanding leaves. SD, stomatal index (SI), and epidermal cell density (ED) were measured on the adaxial and abaxial surfaces of fully expanded upper leaves. SD and SI decreased with increasing lower leaf CO2 concentration (150-780 ppm) at both ambient (1.3-1.6 kPa) and low (0.7-1.0 kPa) D. SD and SI at low D were generally higher than at ambient D. By contrast, ED was relatively insensitive to both vapour pressure and CO2 concentration. When lower leaves were shaded, upper leaf SD, SI, and ED decreased but did not change with varying CO2 concentration. These results suggest that epidermal cell development and stomatal development are regulated by different physiological mechanisms. SI of the upper leaves was positively and highly correlated (r2>0.84) with the stomatal conductance of the lower leaves independent of their net photosynthesis and transpiration rates, suggesting that the stomatal conductance of mature leaves has a regulatory effect on the stomatal development of expanding leaves.

Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm.

Metabolic pathways sequestered within the leucoplast of developing oilseeds ensure a balanced supply of substrates and cofactors for fatty acid biosynthesis. NADP-dependent malic enzyme (NADP-ME) may be important in supplying both carbon and NADPH for fatty acid biosynthesis in the developing endosperm of the oilseed Ricinus communis. NADP-ME was purified 5160-fold to a specific activity of 18.2 U/mg protein. NADP-ME is a homotetramer with a native mass of 254 kDa and a subunit size of approximately 63 kDa. Effectors of castor NADP-ME are typical of the NADP-malic enzymes, with the exception of acetyl-CoA and its derivatives, which were found to act as activators. This is consistent with a regulatory role for these molecules during fatty acid biosynthesis in vivo. NADP-ME was found to have maximal activity at stage 7 of endosperm development, coincident with maximal lipid accumulation.

In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum.

Previously, we described two distinct classes of phosphoenolpyruvate carboxylase (PEPC) isoforms in the green alga Selenastrum minutum. Class 1 PEPC (PEPC1) is a homotetramer composed of 102 kDa subunits (p102), whereas Class 2 PEPCs exist as three large protein complexes (PEPC2-PEPC4) containing varying proportions of structurally dissimilar p102 and 130 kDa (p130) PEPC catalytic subunits. In the current study, a p102 calcium-independent protein kinase was shown to co-purify with PEPC1, but not PEPC2. However, the p130 subunit of PEPC2 was phosphorylated in vitro during its incubation in the presence of [gamma-(32)P]ATP and a clarified algal extract. Treatment of purified PEPC2 with protein phosphatase 2A(2) increased its apparent M(r) as judged by Superose 6 gel filtration chromatography. The presence of the protein phosphatase inhibitors NaF and microcystin-LR throughout PEPC purification significantly influenced the activity and structural organization of Class 2, but not Class 1, PEPC isoforms. The results are consistent with the notion that under the culture conditions employed: (i) Class 1 and Class 2 PEPC isoforms exist in vivo mainly in their dephosphorylated and phosphorylated forms, respectively, and (ii) phosphorylation of Class 2 PEPCs leads to a significant reduction in their activity and native M(r). We propose that protein kinase-mediated phosphorylation is involved in the control and structural organization of green algal PEPC.

A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.