The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase

Abstract

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.

Figure 1

CO₂ assimilation rates of low-light-grown wild-type tobacco plants (▪) and transgenic tobacco plants with a variety of Cyt b/f contents (□) (A and C), and greenhouse-grown wild-type (●) and transgenic tobacco plants with different activities of GAPDH (○) (B and D) were measured using the combined gas exchange and rapid-kill procedure described by Badger et al. (1984). Measurements were conducted at an irradiance of 1,000 μmol quanta m⁻² s⁻¹, a CO₂ partial pressure of 350 μbar or 700 μbar, 21% (v/v) O₂, and a leaf temperature of 25°C. Leaves were kept under these conditions for at least 40 min before the CO₂ assimilation rate was recorded and a leaf disc was rapidly freeze-clamped.

Figure 2

Whole-leaf ATP (A and B) and ADP (C and D) concentrations, the sum of ATP + ADP (E and F), and the ATP/ADP ratio (G and H) in leaves of wild-type, anti-b/f, and anti-GAPDH tobacco plants measured at 350 μbar CO₂. Leaves were freeze-clamped after the gas-exchange measurements at 350 μbar CO₂ shown in Figure 1. Symbols are as in Figure 1. These data are summarized in Table I and compared with similar measurements at 700 μbar CO₂.

Figure 3

NADP-MDH activation levels in the leaves of wild-type, anti-b/f, and anti-GAPDH tobacco plants measured at 350 μbar CO₂. Symbols, measuring conditions, and sampling were as in Figures 1 and 2. The mean total activities were 11.6 ± 0.04 and 10.4 ± 0.15 μmol m⁻² s⁻¹, respectively, for anti-b/f and their wild-type plants and 15.1 ± 0.2 and 18.5 ± 0.3 μmol m⁻² s⁻¹, respectively, for anti-GAPDH and their wild-type plants.

Figure 4

Carbamylation state of Rubisco (A and B), initial in vitro activity of Rubisco per carbamylated (carb.) Rubisco sites (C and D), and total in vitro activity of Rubisco per total Rubisco sites (E and F) in leaves of wild-type, anti-b/f, and anti-GAPDH tobacco plants. Leaves were freeze-clamped after the gas-exchange measurements at 350 μbar CO₂ (shown in Fig. 1). The activity results (C–F) are expressed as a percentage of the average wild-type activities; mean values are given in Table II, which compares them with analog values measured at 700 μbar. The symbols are as in Figure 1.

Figure 5

Ratio between RuBP and Rubisco sites (A and B) and the gross CO₂ assimilation rate per carbamylated (Carb.) Rubisco sites (C and D) and the relationship between gross CO₂ assimilation rate per carbamylated Rubisco site and the ratio between RuBP and Rubisco sites (E and F) in leaves of wild-type, anti-b/f, and anti-GAPDH tobacco plants. The gross CO₂ assimilation rates were calculated as A + R_d, where A is the CO₂ assimilation at 350 μbar CO₂ and R_d the dark respiration rate. Leaves were freeze-clamped after the gas-exchange measurements at 350 μbar CO₂ shown in Figure 1. The symbols are as in Figure 1.