Induction of a C(4)-like mechanism of CO(2) fixation in Egeria densa, a submersed aquatic species

Abstract

The expression of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) in Egeria densa leaves was studied under low temperature and light (LTL) following incubation under high temperature and light (HTL), conditions previously shown to induce high and low CO(2) compensation points, respectively. Transfer from LTL to HTL conditions induced increases in the activities and amounts of both enzymes. One NADP-ME isoform was observed in induced and uninduced samples. Two isoforms of PEPC were expressed, with the lower M(r) isoform being induced by HTL. NADP-ME showed properties similar to those of the isoform in C(3) species. The inducible PEPC isoform has a low K(m) for both substrates. PEPC kinetic and regulatory properties (V(max) and K(m) for phosphoenolpyruvate, and I(50) for L-malate) are different in samples taken in the dark from those in the light, indicating that some modification of PEPC may be occurring during the day. Finally, abscisic acid induced the expression of PEPC and NADP-ME in a manner similar to temperature induction, except that the activities of both PEPC isoforms were increased. A different signaling system may exist in this species in response to high temperature or abscisic acid, both of which induce changes in photosynthetic metabolism.

Figure 1

Effect of high temperature and high light exposure on the specific activity of NADP-ME (A) and PEPC (B) in E. densa. An average of the results obtained from the different repetitions is indicated. se values were less than 5% in all cases.

Figure 2

Effect of high temperature and high light exposure on the content of NADP-ME and PEPC in E. densa. Western-blot analysis of protein samples was performed with antibodies against NADP-ME (A) or PEPC (B). Thirty micrograms of total soluble protein was loaded in each lane. Lane 1, Control, d 0; lane 2, induction for 3 d; lane 3, induction for 15 d.

Figure 3

Two-dimensional western blots of total protein extracts (150 μg) from uninduced (A) and 23-d high-temperature induced (B) E. densa leaves. The membranes were treated with purified anti-Amaranthus viridis PEPC antibodies. The molecular masses of the immunoreactive bands are shown on the left.

Figure 4

Two-dimensional western blots of total protein extracts (150 μg) from uninduced (A) and 23-d high temperature induced (B) E. densa leaves. The membranes were treated with purified anti-maize 62-kD NADP-ME antibody. The molecular masses of the immunoreactive bands are shown on the left.

Figure 5

Western blot of protein samples (30 μg in A and B; 5 μg in C) from supernatant (lane 1) and chloroplast (lane 2) revealed with antibodies raised against maize NADP-ME (A), Amaranthus viridis PEPC (B), and spinach Rubisco large subunit (C). The calculated molecular masses of the immunoreactive proteins are indicated.

Figure 6

Coomassie Blue staining of molecular mass markers (lane 1) and the purified NADP-ME from E. densa (5 μg; lane 2). The molecular masses of the markers are indicated.

Figure 7

Effect of ABA on the specific activity and content of NADP-ME and PEPC in E. densa at 18°C. A, The specific activity of each sample is presented relative to the specific activity measured in control samples taken from untreated plants. An average of the results obtained from the different repetitions is indicated. se values were less than 5% in all cases. B and C, Western-blot analysis of protein samples was performed with antibodies against NADP-ME (B) or PEPC (C). Lane 1, D 0; lane 2, 3 d without ABA; lane 3, 3 d with ABA; lane 4, 10 d without ABA; lane 5, 10 d with ABA; lane 6, 23 d without ABA; lane 7, 23 d with ABA. Thirty micrograms of total soluble protein was loaded in each lane.